Methods and systems for modulating hormones and related methods, agents and compositions

ABSTRACT

Provided herein are bitter taste receptor ligands, related agents, combinations, compositions, methods and systems for modulating release of a metabolic hormone in vitro or in vivo from cells of the GI tract of an individual.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. Provisional Application entitled“Receptors, agents, treatment of diseases and conditions” Ser. No.61/397,940, filed on Jun. 17, 2010, the disclosure of which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT GRANT

The United States Government has rights in this invention pursuant toContract No. between the National Center for Complementary andAlternative Medicine (NCCAM) (1 P01 AT003960).

FIELD

The present disclosure relates to methods and systems for modulatinghormones and related methods, ligands, agents and compositions.

BACKGROUND

Hormones are chemical substances often identified as mediators,typically released by a cell or a gland in one part of an organism toact as a chemical messenger to other parts of the organism.

Various biological processes and in particular metabolic processes areassociated to the release of hormones in an organism. In particularvarious metabolic hormones (e.g. peptide based hormones) affect andregulate the metabolism networks of cells and/or organs in anindividual.

However, controlling hormones production and in particular modulation ofhormone release in connection with treatment of various conditions inthe individual has been challenging.

SUMMARY

Provided herein, are methods, systems and compositions which allow inseveral embodiments, modulating the release of metabolic hormones andrelated biological processes, identifying ligands capable of performingthat modulation and controlling the modulation. In particular providedherein are agents, compositions methods and systems for modulatingrelease of metabolic hormones and controlling related biologicalprocess, including agents, compositions methods and systems that aresuitable for treating metabolic conditions.

According to a first aspect, methods, systems and compositions formodulating release of a metabolic hormone and a related biologicalprocess in an individual are described. The method comprisesadministering to the individual one or more GI bitter taste receptorligands selected from PTU, PTC, denatonium benzoate, Glycyrrhizic acidamomonium salt, Epigallocatechin gallate, Hyperforin, Berberinechloride, Coptisine Chloride Allylsulfide, Rottlerin, Curcumin, Ellagicacid, Embelin, and/or a derivative thereof, in an effective amount toallow binding to the one or more GI bitter taste receptors in theindividual, the binding resulting in modulating the release of themetabolic hormone and related biological process, the metabolic hormoneselected from the group consisting of GLP-1, PYY and CCK. The systemcomprises at least two of one or more GI bitter taste receptor ligandsfor simultaneous combined or sequential use in the method for modulatinghormone release herein described. The composition comprises one or moreligands able to bind one or more target GI bitter taste receptor uponadministering to the individual together with a suitable vehicle.

According to a second aspect, a bitter taste agent for modulating atarget bitter taste receptor, and in particular a GI bitter tastereceptor, in an individual, and related methods, systems andcompositions are described. The bitter taste agents are based on acombination of a bitter taste receptor ligand and a complementarymolecule configured for interfering with systemic absorption and releaseof the bitter taste receptor ligand. In particular, the bitter tasteagent can comprise a bitter taste receptor ligand conjugated with acomplementary molecule wherein the ligand has a first portion and asecond portion. In the bitter taste agent, the first portion of thebitter taste receptor ligand is active with respect to binding of bittertaste ligand to the bitter taste receptor and the second portion ispassive with respect to binding of bitter taste ligand to the bittertaste receptor. In the bitter taste agent, the complementary molecule isattached to the ligand in the second portion of the ligand and theresulting bitter taste agent is configured to present the first portionfor binding to the bitter taste receptor. The method comprisesadministering an effective amount of the bitter taste agent to theindividual. The composition comprises one or more bitter taste receptorligands and at least one complementary molecule. The system comprises atleast two of one or more bitter taste receptor ligands and acomplementary molecule for simultaneous combined or sequential use inproviding a bitter taste agent and/or in the method to modulate hormonerelease herein described.

According to third aspect, a method and systems to identify a biologicalresponse associated to activation of a bitter tastant receptor in acell, and in particular a cell of the GI tract, are described. Themethod comprises contacting the cell with a bitter tastant receptorligand selected from PTU, PTC, denatonium benzoate, Glycyrrhizic acidamomonium salt, Epigallocatechin gallate, Hyperforin, Berberinechloride, Coptisine Chloride Allylsulfide, Rottlerin, Curcumin, Ellagicacid, Embelin, and/or a derivative thereof to allow binding of theligand to the bitter tastant receptor and detecting the biologicalresponse in the cell following the contacting. In some embodiments themethod further comprises comparing the detected biological response to areference biological response to characterize the biological response.The system comprises at least two of one or more bitter taste receptorligands and a cell, for simultaneous combined or sequential use in themethod to identify a biological response herein described.

According to a fourth aspect, methods, systems and compositions forscreening candidate ligands to identify a GI bitter taste receptorligand capable of modulating release of metabolic hormones associated tothe GI system, which in some embodiments is performed based on thespecific locations of a target cell in the GI system and specific bitterreceptors expressed by the target cell. The method comprises identifyinga bitter taste receptor that is expressed in the target cell in the GIsystem, predicting the structure of the GI bitter tastant receptor,identifying a candidate ligand that binds to this receptor based on thepredicted structure, testing the ligand in a bitter taste receptoractivation assay, and then testing the effect of that ligand on themodulation of at least one of the metabolic hormones associated to theGI system.

According to a fifth aspect, a method to modulate a hormone release froma cell comprising inducing a specific conformation of a GI bittertastant receptor through binding of a GI bitter taste receptor ligand,the specific conformation being at least one of a plurality of activeconformations of the bitter tastant receptor, detecting increase ordecrease of a hormone release from a cell following the inducing, andmodulating said increase or decrease through action of the GI bittertaste receptor ligand. In some embodiment, the inducing is preceded byidentifying the GI bitter taste receptor ligand.

According to a sixth aspect, bitter taste receptor ligands, agents, andrelated methods and systems to treat or prevent in an individual acondition associated with a metabolic hormone are described. The methodcomprises administering to the individual one or more GI bitter tastereceptor ligands selected from PTU, PTC, denatonium benzoate or aderivative thereof, in a therapeutically effective amount to modulatemetabolic hormone GLP-1, PYY and/or CCK. The system comprises at leasttwo of a bitter taste receptor ligands or agents herein described forsimultaneous combined or sequential use in the method to treat orprevent in an individual a conditions associated with a metabolichormone herein described.

The methods, systems and compositions herein described allow in severalembodiments modulating the activity of GI bitter taste receptors,modulating secretion and systemic release of metabolic hormones andmodulating related metabolic conditions as well as other biologicalprocesses, including treating metabolic diseases.

The methods and compositions herein described can be used in connectionwith applications wherein modulating activity of GI and intestinalbitter taste receptors is desired, including but not limited to medicalapplication, biological analysis, food processing, taste/flavormodulation, nutrition, nutraceutical applications, and diagnosticsincluding but not limited to clinical applications.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description andexamples section, serve to explain the principles and implementations ofthe disclosure.

FIG. 1 shows sequence alignment against the β1 Adrenergic Receptor(hβ1AR) for TAS2R38 bitter taste receptor variants hTAS2R38_(PAV),hTAS2R38_(AAI), hTAS2R38_(PVV) (taster) and hTAS2R38_(AVI) (nontaster).

FIG. 2 shows the coordinate system used to describe the orientation ofthe seven helices in a GPCR bundle. Double arrows connect nearestneighbor helix pairs that are sampled independently in the BiHelixprocedure. The BiHelix procedure is highlighted using helices 1 and 2 toshow that when the conformations for this helix pair are sampled, otherhelices are not present.

FIG. 3 shows the molecular dynamics simulation box of TAS2R38 bitterreceptor with lipid and water. The EC region is at the top.

FIG. 4 shows predicted 3D structures of bitter taste receptorshTAS2R38_(PAV) (a), hTAS2R38_(AVI) (b), hTAS2R38_(AAI) (c),hTAS2R38_(PVV) (d) from SuperComBiHelix. (Residues forming interhelicalH-bonds are highlighted here)

FIG. 5 shows Predicted 3D structures of bitter taste receptorshTAS2R38_(PAV) (a) and hTAS2R38_(AVI) (b) after 10 ns MD with lipid andwater. (Residues forming interhelical H-bonds are highlighted here andthey are stable during 10 ns MD)

FIG. 6 shows the stability of the predicted hydrogen bonds (HB) inbitter taste receptors over 10 ns MD with full lipid and water. (a)W108-A262 in hTAS2R38_(PAV), (b) Y199-W108 in hTAS2R38_(PAV), (c)Y199-A266 in hTAS2R38_(AVI), (d) W108-A261 in hTAS2R38_(AVI).

FIG. 7 shows Rmsd evolution of each helix of hTAS2R38_(PAV)(a) andhTAS2R38_(AVI)(b) during 10 ns MD (the reference is the last frame)

FIG. 8 shows predicted binding sites of agonists in bitter tastereceptors. (a) PTC in hTAS2R38_(PAV), (b) PTU in hTAS2R38_(PAV), (c) PTCin hTAS2R38_(AVI), (d) PTU in hTAS2R38_(AVI).

FIG. 9 shows the final binding sites of PTC in hTAS2R38_(PAV) (a) andhTAS2R38_(AVI) (b) after 10 ns MD with lipid and water. The essentialelements of the binding mode are retained but additional favorableinteractions are found.

FIG. 10 shows the H-bond distances bonding PTC to hTAS2R38_(PAV) andhTAS2R38_(AVI) during the 10 ns molecular dynamics with lipid and water.(a) PTC-A262 in hTAS2R38_(PAV); (b) PTC-Y199 in hTAS2R38_(PAV); (c)PTC-C198 in hTAS2R38_(AVI); (d) PTC-water in hTAS2R38_(AVI). Note inparticular the formation of the last three hydrogen bonds (b, c and d)not present in the original predicted binding site.

FIG. 11 shows a chemical formula of PTU, an agent according to anembodiment herein described. In the illustration of FIG. 1, the sevencarbon atoms are denoted by the numbers 1 through 7. The sulfur,nitrogen and oxygen atoms are denoted S, N, and O, respectively.

FIG. 12 shows PTU attached to a complementary molecule (R).

FIG. 13 shows PTU functionalized with a carboxylic acid group or anazide to ease linking to a complementary molecule like cellulose or PEG.

FIG. 14 shows Examples of R groups that can be attached to PTU: A.Cellulose; B. PEG.

FIG. 15 shows the atomic structure of PTU as an agent according to anembodiment using an alternative notation, denoting the nitrogen, sulfur,and oxygen atoms, respectively, as N, S, and O.

FIG. 16 shows the atomic structure of PTU-cellulose.

FIG. 17 shows the three dimensional view of the atomic structure ofPTU-cellulose from a first angle.

FIG. 18 shows the three dimensional view of the atomic structure ofPTU-cellulose from a second angle.

FIG. 19 shows the TM helix predictions for hTAS2R47 including thehydrophobic centers for each helix, which are positioned to lie on aplane passing through the middle of the lipid bilayer (Left panel) andthe predicted structure (right panel).

FIG. 20 shows ligand binding sites for Denatonium benzoate, DD2, 4NS,and 6NS complexed to hTAS2R47.

FIG. 21 shows the TM regions for rat and human TAS2R38 taste receptors.

FIG. 22 shows the predicted binding mode for the new pyrimidineantagonist with human DP receptor.

FIG. 23 shows modified pyrimidine compounds, all based on molecule a,shown in FIG. 22.

FIG. 24A shows GLP-1 release from endocrine STC-5 cells triggered by PTCat various concentrations. The X-axis indicates the concentration of PTCused in cell incubation in mM; the Y-axis shows the amount of GLP-1release in pM. Each bar represents an average value obtained fromduplicates.

FIG. 24B shows GLP-1 release from endocrine STC-5 cells triggered by PTCat various concentrations. The X-axis indicates the concentration of PTCused in cell incubation in mM; the Y-axis shows the amount of GLP-1release in pM. Each bar represents an average value obtained fromduplicates.

DETAILED DESCRIPTION

Described herein are methods, systems, and compositions for modulatingrelease of metabolic hormones, related metabolic conditions and otherrelated biological processes, including treating metabolic diseases inan individual.

The term “modulate” or “modulation” as used herein with respect to anactivity of cell membrane receptors and/or a quantifiable biologicalevent, such as release of hormones, indicates the process of interferingwith the activity or biological event. Examples of interfering compriseincreasing, decreasing or maintaining the activity and/or quantifiableevent. For example modulation of the activity of a cell receptor such asGPCRs can be performed by increasing the activity of the GPCR byconverting the GPCR into one of the active conformations which can beperformed for example following interaction of a ligand with a GPCR oreven a single mutation in a GPCR. Analogously, maintaining or decreasingactivity can be performed respectively by maintaining or converting theGPCR into the inactive conformation, which can also be performed throughinteraction of a ligand or various mutations of the GPCRS. Similarly,increasing, decreasing or maintaining of a biological event such asrelease of a biological hormone, can be performed by activation ordeactivation of various intercellular and/or intracellular pathways thatresult from activation of a GPCR, which can performed by operating onthe GPCRs activity. Detection of a modulating activity can be performedfor example through detection of changes in basal activity and/or basalquantifiable event, for example biological and/or chemical indicatorsassociated to the activity and/or the event to be modulated. A skilledperson is able to identify the proper biological and/or chemicalindicators associated to the event of choice using technique and methodsknown to a skilled person and/or identifiable upon reading of thepresent disclosure.

The term “hormones” as used herein indicates a chemical substance oftenidentified as mediator, which is typically released by a cell or a glandin one part of an organism to act as a chemical messenger to other partsof the organism. Exemplary hormones comprise endocrine hormones, whichare released directly into the bloodstream, and exocrine hormones (orectohormones), which are secreted directly into a duct, and, from theduct, they flow either into the bloodstream or from cell to cell bydiffusion in a process known as paracrine signaling. Hormones areproduced by various multicellular organisms and in particularvertebrates. In particular, vertebrate hormones can be categorized inthree chemical classes: Peptide hormones, Lipid and phospholipid-derivedhormones and Monoamines. Peptide hormones consist of chains of aminoacids. Examples of peptide hormones include insulin and growth hormone.Lipid and phospholipid-derived hormones derive from lipids such aslinoleic acid and arachidonic acid and phospholipids. The main classesare the steroid hormones that derive from cholesterol and theeicosanoids. Examples of steroid hormones are testosterone and cortisol.Monoamines derived from aromatic such as phenylalanine, tyrosine, andtryptophan by the action of aromatic amino acid decarboxylase enzymes.Examples of monoamines are thyroxine and adrenaline. For the purposes ofthe present application, hormones will be peptides, lipid andphospholipid-derived hormones and monoamines that are released fromendocrine cells of the gastrointestinal tract including the pancreas.

The term “metabolic hormone” as used herein indicates hormones that arereleased by the endocrine system to regulate the metabolism networks ofcells and organs in an individual. In particular, metabolic hormones canbe peptide based hormones that affect and regulate the metabolismnetworks of cells and/or organs in an individual. Exemplary metabolichormones comprise cholecystokinin (CCK), glucagon like peptide-1 (GLP-1)and peptide Tyrosine Tyrosine (PYY).

In an embodiment, modulating release of a metabolic hormone and arelated biological process thereof in an individual can be performed byadministering to the individual an effective amount of one or moreligands capable of affecting conformational change of one or more targetGI bitter taste receptor in the individual.

The term “bitter taste receptor” or “bitter tastant receptor” indicatesthe family of mammalian taste receptors that detect the sensation ofbitterness, and comprise a distinct subfamily of G protein-coupledreceptors (GPCRs) that doesn't share any homology to other GPCRs. Inparticular, human bitter taste receptor family (TAS2Rs) comprises of ˜25bitter taste receptors. Being GPCRs these receptors exhibit multipleconformations each associated with different biological response, suchas upregulation or downregulation of one or more intracellular pathways.Identification of the conformation can be performed with methods andtechniques such as the methods described in Examples 1 to 3 of thepresent paper as well as other methods and techniques identifiable by askilled person. Detection of TAS2R in active or inactive conformationscan be performed with methods identifiable by a skilled person whichcomprise methods in silico as well detection of biological response thatis known to be associated with either one of the active or inactiveconformations. For example detection of bitter tastants receptors'activation can be performed by measurement of gustducin, and/ortransducin which are released from an heterotrimeric G protein followingconversion of the TAS2R into active conformation [Wong 1995].

Another exemplary biological response that is associated with theconversion or maintenance of TAS2R into an active conformation isprovided by increase in intracellular Ca²⁺ concentration which typicallywith TAS2R receptor in an active conformation in the cell where the Ca²⁺is detected. Detection of gustducin/transducin can be performed byimunnostaining the cell with antibodies for gustducin/transducin or bydetection of any of the biological processes associated therewith.Detecting changes in intracellular Ca²⁺ concentration can be performedby standard assays for GPCRs and intracellular Ca²⁺ measurements [Tsien2003; Zacharias 2000; Zhang 2002]. Additional biological responsesassociated with TAS2R in an active conformation comprise: i) activationof a phosphodiesterase [Keravis 2010]; ii) alteration of potassium ionchannel activity [Scanziani 2009; Schultz 1998]; iii) cell electricalchanges [Scanziani 2009]; iv) hormone or neurotransmitter release,and/or additional responses as will be understood by a skilled personupon reading of the present disclosure.

The term “GI bitter taste receptor” or “GI bitter tastant receptor” asused herein indicates bitter taste receptors located in agastrointestinal tract (GI tract) of an individual. A gastrointestinaltract can comprise the entire gastrointestinal tract of the individualas well as any portion thereof, such as stomach, small intestine,duodenum, jejunum, ileum, large intestine, separately or in anycombination. Typically, the GI bitter taste receptors are located on theluminal facing surface of endocrine cells placed in the epithelium of GItract; and in the islets of Langerhans of the pancreas. Different subsetof bitter taste receptors are expected to be located into differenttypes of GI cells. An exemplary list of GI cells known or expected topresent bitter tastants receptors is shown in Example 13. In particular,a type of GI cells is known or expected to provide a biological responseassociated to conformations (such as one or more of the activeconformations as well as the inactive conformation) of specific TS2Rswhich is determined by the specific type of cell.

The term “ligand” as used herein indicates is a molecule that isrecognized by a particular receptor and binds the receptor in one ormore binding sites. Examples of ligands include, but are not restrictedto, agonists and antagonists for cell membrane receptors, toxins andvenoms, viral epitopes, hormones, hormone receptors peptides, enzymes,enzyme substrates, co factors, drugs (e.g. opiates, steroids, etc.),lectins, sugars, polynucleotides, nucleic acids, oligosaccharides,proteins, and monoclonal antibodies. Formation of a “ligand receptorpair” as used herein indicates combination of the ligand and receptormolecules through molecular recognition to form a complex, which can bedetected by a variety of ligand receptor binding assays known to askilled person [Lefkowitz 1970; de Jong 2005]. Typically, in a ligandtwo portions can be identified: a first portion that is active withrespect to the binding of the ligand to a corresponding receptor and asecond portion that is passive with respect to said binding andcorresponding formation of a ligand receptor pair. In particular, askilled person will understand and will be able to identify an activeportion of a ligand that is involved in the binding and interact orcause interaction with a corresponding binding site of the receptor anda passive portion that is instead not involved and does not affect saidbinding and formation of a corresponding ligand receptor pair.

In some embodiments herein described, formation of a ligand receptorpair affects the conformation of the bitter taste receptor and inparticular either retains an existing conformation or converts theexisting conformation into a new conformation. For example, in some ofthose embodiments the ligand is an antagonist of the receptor and theformation of the ligand pair receptor complex results into retaining ofthe inactive conformation. In other embodiments the ligand is an agonistof the receptor and formation of the ligand pair receptor complexresults in conversion of the inactive form into one or more of theactive forms of the receptor. TS2R receptor forms are associated in turnwith activation or inactivation of at least one intracellular pathwayresulting in one or more detectable chemical or biological response.

In several embodiments, at least one of the active conformations of abitter tastant receptor results in modulation of release of metabolichormones by endocrine cells where a target receptor is located. Inparticular, bitter taste receptor ligands (BTRL) can either causeactivation of any of the multiple intracellular pathways or preventactivation of any of those intracellular pathways which results in acorresponding modification of the hormone release.

Detection of modulation of a bitter taste GPCR along with its effect onrelease of a specific hormone or neurotransmitter of interest can beperformed by a skilled person in vitro or in vivo by measuringdisplacement of the hormone or neurotransmitter of interest from itsintracellular site to outside the cell in the case of in vitromeasurements or into the blood in the case of in vivo measurements or bydetection of another biological response as herein described.

Detection of release of a specific hormone can be also performed bydetecting release of hormone in extracellular environment and bodilyfluids and in particular in blood using techniques identifiable by askilled person. For example, detection in blood can be performed byseparating a sample blood of an individual into plasma, serum and bloodcell fractions. Using standard radio-immunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA) techniques, a specific hormone is measuredin either the serum or plasma fraction. Typically, both RIA and ELISArequire specific antibodies for each hormone to be measured. Suitableantibodies are available for each hormone from commercial sources andacademic centers.

In some embodiments activation or inactivation of intracellular pathwaycan be specifically associated with bitter sensation as will beunderstood by a skilled person. In those embodiments. In thoseembodiments the ligand able to affect the conformation of the bittertaste receptor typically comprises one or more bitter tastants. The term“bitter tastant” as used herein indicates a substance that is recognizedby bitter taste receptors to elicit sensation of bitterness. Sensationof bitterness can be detected using a reference compound such asquinone. Accordingly, the threshold for stimulation of bitter taste byquinine averages 0.000008 M. The taste thresholds of other bittersubstances are rated relative to quinine. Exemplary bitter tastantsaccording to the current disclosure include but are not limited to6-n-Propylthiouracil (PTU), Phenylthiocarbamide (PTC), Denatoniumbenzoate and some derivatives thereof. Chemical structures of PTC andPTU (also called PROP) are shown below

In other embodiments, binding of a ligand and related active or inactiveconformation of the TAS2R receptor does not involve activation orinactivation of one or more cellular pathways associated with the bittertaste sensation.

In some embodiment the ligand can be a derivative of an existing ligand.The term “derivative” as used herein with reference to a first compound(e.g. PTU), indicates a second compound that is structurally related tothe first compound and is derivable from the first compound by amodification that introduces a feature that is not present in the firstcompound while retaining functional properties of the first compound.Accordingly, a derivative compound of PTU, usually differs from theoriginal compound by modification of the chemical formula that might ormight not be associated with an additional function not present in theoriginal compound. A derivative compound of PTU retains however one ormore functional activities that are herein described in connection withcompound in association with the ability of PTU to bind bitter tastantreceptors. Accordingly, derivatives of a PTU or other BTRL comprise anychemically modified form of the tastant, given that the derivativesretain the ability of binding TAS2R38 converting the corresponding tastereceptor into one or more active conformations. Derivatives of a PTU orother BTRL also comprise any chemically modified form that retain theability to bind TAS2R38 but does not retain the ability to modify theinactive conformation of TAS2R38. In embodiments, where the derivativesmaintain the ability to affect the conformation of the target receptor,chemical modifications can be performed in the passive portion of theligand. In embodiments where the derivatives do not retain the abilityto modify the inactive conformation of TAS2R38, chemical modificationscan be performed on the active as well as passive portion of the ligand.Specific residues to be modified to derive antagonist can be identifiedby modeling the specific receptor to identify ligands capable of bindingTAS2R38. Test the biological response of the ligand to identify theinability of the ligand to activate the TAS2R38 receptor and select theantagonist of TAS2R38. Performing a cavity analysis of the antagonistbinding site to identify the PTU-based derivative binding residues thatare associated with the antagonistic activity on TAS2R38. A skilledperson will be able to identify further antagonist derivatives of PTU bymodifying PTU to preserve the same binding site of the PTU-basedderivative antagonist. Chemical modifications are determined based onthe modeling of tastant binding site in the bitter receptor (e.g.replacing functional group on the ligand to create an additionalinteraction). Exemplary chemical modifications of a tastant according tothe current disclosure that do not affect the ability to activateconformation of the TAS2R38 include modifying the propyl chain in thePTU (PROP) molecule as this group does not interact with the bitterreceptor based on the predicted binding site. Accordingly, in variouscases, based on the tastant's predicted binding site, a core structurefor an agonist derivative can be defined as the part that interacts withthe bitter receptor and hence shouldn't be modified. This will vary foreach different ligand. The nature and function of derivatives isdiscussed later.

An effective amount is the amount that results in a concentration of aligand at a cellular level that allows formation of a receptor bindingcomplex and hormone release. In some embodiments, for example, aneffective amount comprises a concentration of active agents from about 1micromolar to about 1.25 millimolar. Additional amounts known orexpected to be effective for PTC, PTU, Denatonium Benzoate and otherligands herein described comprise from about 0.5 uM to about 1 uM, fromabout 1 uM to about 2.5 uM, from about 2.5 uM to about 5 uM, from about5 uM to about 10 uM, from about 10 uM to about 100 uM, from about 100 uMto about 500 uM, from about 500 uM to about 1000 uM, from about 1 mM toabout 1.25 mM, from about 1.25 mM to about 2.5 mM, from about 2.5 mM toabout 5 mM, from about 5 mM to about 10 mM. Additional effective amountsknown or expected to be effective comprise from about 1-2.5 uM, fromabout 2.6 uM to about 10 uM, from about 11 uM to about 100 uM; fromabout 101 uM to about 1000 uM; from about 1.25 mM to about 10 mM.

In particular an effective amount in several embodiments is known orexpected to be about 2.5 uM. The effective amounts are known or expectedfor the various ligands herein described alone or in combination and inparticular for PTC, PTU, Denatonium Benzoate, Coptisine chloride, Allylsulfide, Rottlerin, Curcumin, Ellagic acid, Embelin and all the otherligands mentioned in the examples.

In various embodiments, the administering of GI bitter taste receptorligands, in particular bitter tastant, and related agents can beperformed through any route of administration that ensures directdelivery to the desired GI tract. e.g. through oral administration routeor other administration route. In particular in some embodiments, theagent is delivered to the lumen of the GI tract and is configured tominimizes systemic transfer of agent in whole or in part from the lumenacross the epithelium of the GI tract and enter the blood. In some ofthose embodiments, minimization of the systemic transfer from the lumenof the GI tract provides action specificity and decreases adverseeffects.

In an embodiment, GI bitter taste receptor ligands, related agents andcompositions comprising the same, can be delivered into the GI orintestine. In particular, in various embodiments GI refers to the wholeGI system, The GI tract in the sense of the present disclosure caninclude all parts of the GI system from the mouth to the anus; and theattached pancreas and liver. Effects of hormones released from the GIsystem will or are expected in turn to have secondary effects onpancreas and liver that are desirable for the full beneficial response.

In particular, in some embodiments GI bitter taste receptors are locatedin GI tract portions upstream of the stomach. The term “upstream” asused herein indicates the portion the mouth of the esophagus. In someembodiments, bitter taste receptors are located in the stomach i.e. anytype of bitter taste receptors that are located in the stomach. In someembodiments, bitter taste receptors are intestinal bitter tastereceptors i.e. any type of bitter taste receptors that are located inthe intestine (both large and small). A distribution of different bittertaste receptors in the GI system is expected. The localization of thesereceptors in the GI system would affect the choice of the agent beingadministered as would be understood by a skilled person upon reading ofthe present disclosure. In particular, the TAS2R and the cellspresenting the TAS2R to be targeted can be selected based on thespecific hormone to be modulated and the modulation effect desired.Location of the selected cell on the GI tract can be identified. Aligand capable to obtain the desired modulation effect in the selectedcell can therefore be administered in the GI tract where the selectedcells are located in a suitable amount and form that ensure delivery inthe selected tract. For example, increase of GLP-1 can be performed byactivating TAS2R38 in cells located in the small and large intestine. Aligand suitable to activate the TAS2R38 in the L cells is PTU. A skilledperson would understand that an effective amount of PTU can be delivereddirectly to the intestine to obtain the desired increase in GLP-1production release from the L cell into the blood A skilled person willalso understand that due to the chemical nature of PTU and the relevantability to be systemically absorbed by the GI epithelium, PTU can bedesirably conjugated with a complementary molecule to minimizeabsorption and maximize half-life in the intestine.

In an embodiment, the GI and intestinal bitter taste receptors comprisebitter taste receptors located on endocrine cells located on the luminalsurface of the GI tract, such as TAS2R38 and TAS2R47. In some of thoseembodiments, activation of the GI bitter taste receptors results in therelease, and consequent systemic distribution, of metabolic hormonesfrom hormone releasing cells, including release from or mediated by saidendocrine cells (which cells may be activated from within the GI tractor intestine to release hormones into the blood). Delivery of bittertastants receptor ligands, combinations and compositions thereof to theGI tract or intestine is also expected to affect said release throughintervening, complementary, and alternative steps and processes.

In some embodiments, the method comprises administering to theindividual one or more GI bitter taste receptor ligands and/or relatedagents capable of activating one or more target GI bitter taste receptorin the individual in an effective amount to modulate release of one ormore metabolic hormones in the individual.

In an embodiment, GI bitter taste receptor ligands, and agents hereindescribed can be delivered to the GI tract to modulate the release ofmetabolic hormones from endocrine cells of the GI tract, such as theCCK-containing I-cells and the GLP-1 and PYY-containing L cells. Inanother embodiment, ligands and agents herein described are expected tobe effective on bitter taste receptor present on neural endings of theGI tract and to modulate nerve response which in turn modulates hormonerelease from endocrine cell in the GI tract.

In an embodiment, GI bitter taste receptor ligands, related agents,compositions, methods and systems are suitable to modulate GLP-1. Insome of these embodiments, the ligand can be PTC, PTU, Glycyrrhizic acidammonium salt, Epigallocatechin gallate, Hyperforin, Berberine chloride,Coptisine Chloride Allyl methyl sulfide, Rottlerin, Curcumin, Ellagicacid, Embelin and/or an agonist derivative thereof. In particular, PTCand PTU, or any of the other ligand applied alone or in combination tothe luminal surface of hormone releasing GLP-1 and PYY-containing L cellwill result in activation of the TAS2R38 receptor on the luminal surfaceof the L-cell resulting in the release of hormones GLP-1 and PYY fromthe hormone containing cells into the blood. In particular, the bittertaste receptor ligands, related agents and combinations thereof can beapplied to the luminal surface of the GI tract at concentrations rangesfrom about 1 uM to about 2.5 uM, from about 2.6 uM to about 10 uM, fromabout 11 uM to about 100 uM; from about 101 uM to about 1000 uM; fromabout 1.25 mM to about 10 mM, with particular reference to systems thatallow delivery to the specific site of location of the TAS2R38 receptoron GLP-1 and PYY-containing L cells in the GI tract. Other ligandscapable to activate TAS2R38 receptors applied to the luminal surface ofthe GI tract at concentrations from about 1 uM to about 2.5 uM, fromabout 2.6 uM to about 10 uM, from about 11 uM to about 100 uM; fromabout 101 uM to about 1000 uM; from about 1.25 mM to about 10 mM, withparticular reference to systems that allow delivery to the specific siteof location of the TAS2R38 receptor on GLP-1 and PYY-containing L cellsin the GI tract are also expected to result in release of GLP-1 and PYYfrom the hormone containing cells into the blood

In an embodiment, GI bitter taste receptor ligands, related agents,compositions, methods and systems are suitable to modulate release ofCCK into the blood. In some of these embodiments, the ligand isdenatonium benzoate or an agonist derivative thereof. These agentsapplied alone or in combination to the luminal surface of hormonereleasing CCK-containing I cell will result in activation of TAS2R47receptor located on the luminal surface of the I cell resulting in therelease of hormone CCK from the hormone containing cells into the blood.In the particular in some embodiments, the agents are applied to theluminal surface of the GI tract at concentrations from about 1-2.5 uM,from about 2.6 uM to about 10 uM, from about 11 uM to about 100 uM; fromabout 101 uM to about 1000 uM, from about 1.25 mM to about 10 mM insystems that allow delivery to the specific site of location of theTAS2R47 receptor on CCK-containing I cells in the GI tract. Otherligands capable of activating TAS2R47 receptors applied to the luminalsurface of the GI tract at concentrations from about 1 uM to about 2.5uM, from about 2.6 uM to about 10 uM, from about 11 uM to about 100 uM;from about 101 uM to about 1000 uM; from about 1.25 mM to about 10 mM,with particular reference to systems that allow delivery to the specificsite of location of the TAS2R47 receptor on CCK-containing I cells inthe GI tract will also result in the release of CCK from the hormonecontaining cells into the blood.

In an embodiment, GI bitter taste receptor ligands compositions, methodsand systems are suitable to modulate PYY. In some of those embodiments,since PYY is expected to be released by L-cells, agents able to modulateGLP-1 are also expected or known to modulate PYY.

In some embodiments, the one or more target GI bitter taste receptorcomprises TAS2R38, and the one or more metabolic hormones comprisesGLP-1 and PYY. In other embodiments, the one or more target GI bittertaste receptor comprises TAS2R47, and the one or more metabolic hormonescomprises CCK.

In some embodiments, the one or more agent comprises a bitter tastant.In particular, in some embodiments, the bitter tastant is selected fromthe group consisting of PTU, PTC, Denatonium benzoate, a derivativethereof and a combination thereof. In particular, both ligands andagents activating TAS2R47 (e.g. DB) and ligands and agents activatingTAS2R38 (e.g. PTU and PTC) can be applied alone or in combination to theluminal surface of the GI tract at concentrations from about 1 uM toabout 2.5 uM, from about 2.6 uM to about 10 uM, from about 11 uM toabout 100 uM, from about 101 uM to about 1000 uM, from about 1.25 mM toabout 10 mM. In some of those embodiments, performed in systems thatallow delivery to the specific site of location of the TAS2R47 receptoror TAS2R38, respectively, administration is known or expected to resultin the release of hormones into the blood. In some embodiments, ligandsand agents that activate TAS2R47, are known or expected to result inrelease of the hormone CCK from 1 cells in the upper part of theintestine into the blood. In an embodiment, agents or ligands thatactivate TAS2R38 are known or expected to result in release of thehormones GLP-1 and PYY from the L cell in the lower part of theintestine the blood.

In more particular, in some embodiment, the bitter tastant is selectedfrom the group consisting PTU, PTC, a derivative thereof and acombination thereof, and the one or more metabolic hormones comprisesGLP-1 and PYY. In other embodiments, the bitter tastant is selected fromthe group consisting of Denatonium benzoate, a derivative thereof and acombination thereof, and the one or more metabolic hormones comprisesCCK. In some embodiments, the combination can being particular deliveredin forms that do not leave the intestinal lumen. In this way, each wouldbe delivered to the proper location in the intestine. In particular, insome of these embodiments, combination can be administered before andduring a meal. Because of the kinetics of movement, the ligands for theI cell will reach the I cell before the ligands for the L cell reach theL cell. Accordingly, an effect of CCK that predominantly slow emptyingof the stomach is expected to occur initially. For example in one caseat about 30 min to about 2 hours after administration of the agents, theligands for the L cell is expected to reach the target cells andinitiate release of GLP-1 and PYY which have effects on gastric emptyinglike CCK but also promote satiety and insulin release. The combinationof effects is expected to be beneficial for both weight control anddiabetes as well as the consequences of these disorders. In someembodiments, a sustained benefit is expected with a combination ofligands and/or agents administered before and with all meals for aprolonged amount of time (e.g. for many months).

In some embodiments, the one or more agents comprises a bitter tastant,the one or more target GI bitter taste receptor comprises TAS2R47located on I cells and TAS2R38 located on L cells, and the one or morehormones comprises CCK, GLP-1 and PYY. In particular, in someembodiments, wherein the one or more agent comprises a bitter tastant,the one or more target GI bitter taste receptor comprises TAS2R38located on L cells, and the one or more hormones comprises PYY, GLP-1located in the L cell. In other embodiments, wherein the one or moreagent comprises a bitter tastant, the one or more target GI bitter tastereceptor comprises TAS2R47 located on I cells, and the one or morehormones comprises CCK located in the I cell. In more particular, insome embodiments, the bitter tastant is selected from the groupconsisting of PTU, PTC, Denatonium benzoate, a derivative thereof and acombination thereof, the one or more target GI bitter taste receptorcomprises TAS2R38 located on L cells, and the one or more hormonescomprises PYY, GLP-1. In other embodiments, the bitter tastant isselected from the group consisting of PTU, PTC, Denatonium benzoate, aderivative thereof and a combination thereof, the one or more target GIbitter taste receptor comprises TAS2R47 located on I cells, and the oneor more hormones comprises CCK.

In some embodiments, the one or more agent is a composition comprisingone or more bitter tastant. In particular, in some embodiments, the oneor more bitter tastant is selected from the group consisting of PTU,PTC, a derivative thereof and a combination thereof, and the one or moremetabolic hormones comprises GLP-1 and PYY. In other embodiments, theone or more bitter tastant is selected from the group consisting ofDenatonium benzoate, a derivative thereof and a combination thereof, andthe one or more metabolic hormones comprises CKK.

In some embodiments, the one or more agent comprises a compositioncomprising one or more bitter tastant. In particular, in someembodiments, the one or more bitter tastant is selected from the groupconsisting of PTU, PTC, Denatonium benzoate, a derivative thereof and acombination thereof. The one or more target bitter taste receptorcomprises TAS2R38 located on L cells, and the one or more hormonescomprises PYY, GLP-1. In other embodiments, the one or more bittertastant is selected from the group consisting of PTU, PTC, Denatonium, aderivative thereof and a combination thereof. The one or more targetbitter taste receptor comprises TAS2R47 located on L cells, and the oneor more hormones comprises CKK.

In some embodiments, the one or more agent comprises a bitter tastantand the administrating is performed by orally administrating the one ormore agent into a GI tract of the individual, the one or more target GIbitter taste receptor comprises TAS2R38 and TAS2R47 and the one or moremetabolic hormone comprises PYY, GLP-1 and CCK, wherein the bittertastant is suitable for oral administration to the GI tract, inparticular, neither the bitter tastant nor its metabolites causesadverse effects delivered in a system that minimizes absorption acrossthe GI tract and distribution systemically. In particular, in someembodiments, the one or more target GI bitter taste receptor comprisesTAS2R38, and the one or more metabolic hormone comprises PYY, GLP-1. Inother embodiments, the one or more target GI bitter taste receptorcomprises TAS2R47, and the one or more metabolic hormone comprises CCK.

In some embodiments, the agent comprises bitter tastant capable oftransiting to and through part or all of the GI tract or intestine, uponoral or other administration

In embodiments, where administration is performed by oral route rectalroute or other route that is suitable to deliver the ligand to thedesired tract of the intestine, ligands or combination thereof can be informulation such that the ligand does not lose its functionality throughdegradation, additive reaction, digestive enzymatic or other process,metabolic or bacterial process, or otherwise, prior to or upon deliveryto the portion or portions of the GI tract or intestine wherein residethe bitter taste receptors to which the ligand is intended to bedelivered.

In some embodiments, the ligand is capable of transiting to and throughpart or all of the GI tract or intestine, upon oral or otheradministration route of administration. In other embodiments, the ligandnaturally has a minimal absorption through the GI tract or is modifiedto minimize absorption across the GI tract. In some embodiments, toprolong the half-life of a ligand in the intestine, the ligand isexpected to be non-degrading and in at least some cases to be resistantor impervious to degradation by the digestive enzymes or other chemicalsand action of the stomach and intestine. A tether if present is to beconnected to the agent in a way and/or at such a point on the agent thatit does not interfere with/inhibit the agent's functioning/binding.

In some embodiments, ligands can be comprised in a composition, providedin combination or otherwise modified for use as or in an agent. Inparticular, in some embodiments, ligands are comprised in compositionsthat are formulated to minimize absorption of the ligand across the GItract

In some embodiments, a ligand or agent or a combination thereof can becomprised in composition that delivers an agent specifically to thevicinity of a cell or part of the intestine, or directly to a specificcell. In particular, in some embodiments, delivery can be performed byencapsulation (including micelle encapsulation), in other embodiment byconjugation to a chain of multipart block co-polymers that target a cellor environment and then release or activate the agent/ligand ‘payload’according to techniques and procedures identifiable by a skilled person

In some embodiments, a ligand can be administered in combination with acomplementary molecule configured for interfering with systemicabsorption of the bitter tastant.

The term “complementary molecule” as used herein indicates a moleculeconfigured to modify distribution of at least one BTRL to minimizeabsorption through GI tract without substantially altering ability ofthe ligand to bind a corresponding TAS2R(s). Exemplary types of thecomplementary molecule according to the current disclosure, includepolymers such as nucleic acid, protein, PEGs, monosaccharide andoligosaccharides (such as cellulose) These molecules are known orexpected to prevent the absorption of BTRLs in the GI system to prolongtheir effect on the hormone release. Exemplary complementary moleculescomprise monosaccharides, oligosaccharides, amino acids, peptides, andpolymers such as cellulose or PEG.

The term “monosaccharide” as used herein indicates the most basic unitsof biologically important carbohydrates. Typically monosaccharides havethe chemical formula C_(x)(H₂O)_(y), where x is at least 3.Monosaccharides can be classified by the number x of carbon atoms theycontain: diose (2) triose (3) tetrose (4), pentose (5), hexose (6),heptose (7), and so on. Monosaccharides can be classified in linearchain monosaccharide, open chain stereoisomers, and cyclic isomers.Examples of monosaccharides include glucose (dextrose), fructose(levulose), galactose, xylose and ribose. Monosaccharides are thebuilding blocks of disaccharides such as sucrose and polysaccharides(such as cellulose and starch). Further, each carbon atom that supportsa hydroxyl group (except for the first and last) is chiral, giving riseto a number of isomeric forms all with the same chemical formula. Forinstance, galactose and glucose are both aldohexoses, but have differentchemical and physical properties.

The term “oligosaccharide” as used herein, indicates a saccharidepolymer containing a small number (typically two to ten) of componentsugars, also known as simple sugars (monosaccharides). Oligosaccharidescan have many functions; for example, they are commonly found on theplasma membrane of animal cells where they can play a role in cell-cellrecognition. In general, they are found either O- or N-linked tocompatible amino acid side-chains in proteins or to lipid moieties (seeglycans).

As used herein the term “amino acid”, “amino acidic monomer”, or “aminoacid residue” refers to any of the twenty naturally occurring aminoacids, non-natural amino acids, and artificial amino acids and includesboth D an L optical isomers. In particular, non-natural amino acidsinclude D-stereoisomers of naturally occurring amino acids (theseincluding useful ligand building blocks because they are not susceptibleto enzymatic degradation). The term “artificial amino acids” indicatemolecules that can be readily coupled together using standard amino acidcoupling chemistry, but with molecular structures that do not resemblethe naturally occurring amino acids. The term “amino acid analog” refersto an amino acid in which one or more individual atoms have beenreplaced, either with a different atom, isotope, or with a differentfunctional group but is otherwise identical to original amino acid fromwhich the analog is derived. All of these amino acids can besynthetically incorporated into a peptide or polypeptide using standardamino acid coupling chemistries. The term “polypeptide” as used hereinincludes polymers comprising one or more monomer, or building blocksother than an amino acid monomer. The terms monomer, subunit, orbuilding blocks indicate chemical compounds that under appropriateconditions can become chemically bonded to another monomer of the sameor different chemical nature to form a polymer. The term “polypeptide”is further intended to comprise a polymer wherein one or more of thebuilding blocks is covalently bound to another by a chemical bond otherthan amide or peptide bond.

The term cellulose as used herein indicates an organic compound with theformula (C₆H₁₀O₅)_(n), a polysaccharide consisting of a linear chain ofseveral hundred to over ten thousand β(1→4) linked D-glucose units.Cellulose is the most common organic compound on Earth. About 33% of allplant matter is cellulose (the cellulose content of cotton is 90% andthat of wood is 40-50%). Methods and techniques for synthesis and usesof cellulose to be administered in a composition and in particular apharmaceutical composition for individuals are identifiable by a skilledperson.

The term “Polyethylene glycol” or “PEG” as used herein indicates apolyether compound. Exemplary PEG molecules comprise PEG, PEO, or POEwhich refer to an oligomer or polymer of ethylene oxide. The three namesare chemically synonymous, but historically PEG has tended to refer tooligomers and polymers with a molecular mass below 20,000 g/mol, PEO topolymers with a molecular mass above 20,000 g/mol, and POE to a polymerof any molecular mass. PEG and PEO are liquids or low-melting solids,depending on their molecular weights. PEGs are prepared bypolymerization of ethylene oxide and are commercially available over awide range of molecular weights from 300 g/mol to 10,000,000 g/mol. Insome embodiments, PEG suitable to be used as a complementary molecule isPEG 10,000 [Kerckhoffs, 2010].

In an embodiment, the ligand can be comprised in composition togetherwith a complementary molecule configured to minimize absorption of theligand in the GI system. In an embodiment, a composition can comprisemore than one agent and/or ligands as well as suitable vehicle oradditives. Exemplary compositions comprise PTU-Cellulose or PTU PEG Askilled person can identify complementary molecule suitable to be notabsorbable across GI tract, and linking or encapsulating to meet thecriteria.

In some embodiments, the bitter tastant receptor ligand can beconjugated with the complementary molecule. The term “conjugate” or“conjugation” as used herein indicates association of at least twomolecules into a complex through covalent binding. The term “covalentbinding” as used herein indicates a process of formation of a chemicalbonding that is characterized by sharing of pairs of electrons betweenatoms, known as the covalent bond. Covalent bonding indicates a stablebalance of attractive and repulsive forces between atoms when the atomsshare their electrons, and includes many kinds of interaction, includingσ-bonding, π-bonding, metal to metal bonding, agostic interactions, andthree-center two-electron bonds. In several embodiments, the conjugatinggroup is expected to be a reactive group like carboxylic acid and azideshown in FIG. 13, which facilitates the attachment of most types ofcomplementary molecules. In particular, in some embodiments, conjugationcan be performed by direct attachment of the bitter taste receptorligand with the complementary molecule. In some embodiment, thecomplementary molecule can be conjugated by indirect attachment whereinthe bitter taste receptor ligand is covalently bound to thecomplementary molecule through one or more additional molecule (e.g. alinker enabling or facilitating attachment of the ligand to thecomplementary molecule). In particular, conjugation is performed so thatthe bitter taste receptor ligand in the resulting agent comprising theligand linked to the complementary molecule is presented for binding toa corresponding GI bitter taste receptor.

The term “present” as used herein with reference to a compound orfunctional group indicates attachment performed to maintain the chemicalreactivity of the compound or functional group as attached. Accordingly,a functional group presented on a ligand, is able to perform under theappropriate conditions the one or more chemical reactions thatchemically characterize the functional group.

The term “attach” or “attached” as used herein, refers to connecting oruniting by a bond, link, force or tie in order to keep two or morecomponents together, which encompasses either direct or indirectattachment where, for example, a first molecule is directly bound to asecond molecule or material, or one or more intermediate molecules aredisposed between the first molecule and the second molecule or material.

The term “functional group” as used herein indicates specific groups ofatoms within a molecular structure that are responsible for thecharacteristic chemical reactions of that structure. Exemplaryfunctional groups include hydrocarbons, groups containing halogen,groups containing oxygen, groups containing nitrogen and groupscontaining phosphorus and sulfur all identifiable by a skilled person.In particular, functional groups in the sense of the present disclosureinclude a carboxylic acid, amine, triarylphosphine, azide, acetylene,sulfonyl azide, thio acid and aldehyde. In particular, for example, thefirst functional group and the second functional group can be selectedto comprise the following binding partners: carboxylic acid group andamine group, azide and acetylene groups, azide and triarylphosphinegroup, sulfonyl azide and thio acid, and aldehyde and primary amine.Additional functional groups can be identified by a skilled person uponreading of the present disclosure. As used herein, the term“corresponding functional group” refers to a functional group that canreact to another functional group. Thus, functional groups that canreact with each other can be referred to as corresponding functionalgroups.

In some embodiments, the conjugated bitter tastant or the compositioncomprises PTU. In some of those embodiments, PTU is covalently bond tothe complementary molecule. In particular, in some embodiments, a singlehydrogen atom of PTU as shown in FIG. 11 is replaced by an OH functionalgroup that is shared by, linked to, incorporated in, or incorporated byboth PTU and the complementary molecule.

In particular, in some embodiments, the complementary molecule is anucleic acid. In some embodiments, the complementary molecule is aprotein. In some embodiments, the complementary molecule is amonosaccharide. In some embodiments, the complementary molecule is anoligosaccharide.

In some embodiments, the composition comprises PTU and at least twocomplementary molecules, wherein at least one complementary molecule iscovalently bonded to PTU, and at least one other complementary moleculeis not covalently bound to the ligand.

In particular, in some embodiments, the covalent bonding between the atleast one complementary molecule and PTU is through replacement of asingle hydrogen atom of PTU as shown in FIG. 11 by a carboxylic or azidefunctional group (FIG. 13) that is shared by, linked to, incorporatedin, or incorporated by both PTU and the complementary molecule.

In some embodiments, PTU can be modified, composed, encapsulated orotherwise prepared for use in delivery to the GI tract or intestine, orfor modulation of GI or intestinal bitter taste receptors and inparticular to activate or modulate GI or intestinal bitter tastereceptor TAS2R38 or other GI or intestinal bitter taste receptors.

In an embodiment, activating a target bitter tastant receptor in anindividual are described. The method comprises administering to theindividual an effective amount of one or more agents selected from thegroup consisting of PTU, PTC, denatonium, a derivative thereof and acombination thereof. The system comprises at least two agents selectedfrom the same group. The composition comprises one or more agentselected from the same group.

In some embodiments, the conjugated bitter tastant agent comprises oneor more compound selected from the group consisting berberine chloride,cyanidine chloride, coptisine chloride, any functional derivativethereof and any combination thereof. In some embodiments, the endocrinecells are I-cells. In other embodiments, the endocrine cells areL-cells. In some embodiments, Berberine chloride, cyanidine chloride,coptisine chloride, any functional derivative thereof and anycombination thereof can also be provided in methods, compositions andsystems herein described as simple ligands. In some of thoseembodiments, ligands and relate agents can be used in applications fornutraceutical uses.

In an embodiment, use of bitter tastant receptor ligands and relatedcompositions, and to the modulation of GI and intestinal bitter tastereceptors, can be directed to modulate the release of GLP-1, PYY, CCKand other metabolic hormones, including, but not limited to, metabolichormones that modulate Metabolic and Related Diseases and Conditions andbiological processes relating to the foregoing. GLP-1, PYY, CCK, andother metabolic hormones are known to be released into the blood bygastrointestinal constituents in the lumen of the GI tract, and areknown to regulate functions such as gastric emptying, satiety, insulinsecretion, lipid metabolism, and other metabolic-related processes.

The term “condition” as used herein indicates a physical status of thebody of an individual (as a whole or as one or more of its parts), thatdoes not conform to a standard physical status associated to a state ofcomplete physical, mental and social well-being for the individual.Conditions herein described include but are not limited disorders anddiseases wherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms.

The wording “associated to” as used herein with reference to two itemsindicates a relation between the two items such that the occurrence of afirst item is accompanied by the occurrence of the second item, whichincludes but is not limited to a cause-effect relation andsign/symptoms-disease relation.

Accordingly, the term “metabolic condition”, “metabolic disease” or“metabolic related condition” as used herein indicates a condition ordisease related to one or more metabolic processes and/or dysfunction,including a condition or disease treatable through modulation of releaseof metabolic hormones. Exemplary metabolic condition or diseasesaccording to the current disclosure include but are not limited toobesity, diabetes, liver diseases and cardiovascular diseases. Becauseobesity and diabetes are associated with an increased risk of severalcancers, treatment of obesity and diabetes will have a beneficial effecton these cancers as well as metabolic syndrome.

The term “biological process” as used herein in the context ofbiological processes related to a metabolic condition or disease asdescribed above, refers to a biological process that influences and/orcauses the metabolic condition or disease as described above, includingbut not limited to gastric emptying, satiety, insulin secretion, lipidmetabolism.

According to several embodiments, bitter taste receptor ligands, andrelated composition methods and systems are herein described to treat orprevent in an individual a condition associated to a metabolic hormone

The term “treatment” as used herein indicates any activity that is partof a medical care for or deals with a condition medically or surgically.

The term “prevention” as used herein indicates any activity, whichreduces the burden of mortality or morbidity from a condition in anindividual. This takes place at primary, secondary and tertiaryprevention levels, wherein: a) primary prevention avoids the developmentof a disease; b) secondary prevention activities are aimed at earlydisease treatment, thereby increasing opportunities for interventions toprevent progression of the disease and emergence of symptoms; and c)tertiary prevention reduces the negative impact of an alreadyestablished disease by restoring function and reducing disease-relatedcomplications.

The term “individual” as used herein in the context of administrating aagent includes a single biological organism, including but not limitedto, animals and in particular higher animals and in particularvertebrates such as mammals and in particular human beings.

An effective amount and in particular a therapeutically effective amountof a bitter taste receptor ligand or agent alone or in combinationcomprise from about 1 micromolar to about 1.25 millimolar. Additionalamounts known or expected to be effective for PTC, PTU, DenatoniumBenzoate and other ligands herein described comprise from about 0.5 uMto about 1 uM, from about 1 uM to about 2.5 uM, from about 2.5 uM toabout 5 uM, from about 5 uM to about 10 uM, from about 10 uM to about100 uM, from about 100 uM to about 500 uM, from about 500 uM to about1000 uM, from about 1 mM to about 1.25 mM, from about 1.25 mM to about2.5 mM, from about 2.5 mM to about 5 mM, from about 5 mM to about 10 mM.Additional effective amounts known or expected to be effective comprisefrom about 1-2.5 uM, from about 2.6 uM to about 10 uM, from about 11 uMto about 100 uM; from about 101 uM to about 1000 uM; from about 1.25 mMto about 10 mM.

In an embodiment, diseases and conditions that can be treated orprevented through release or modulation of release of GLP-1, PYY, CCK orother metabolic hormones include, but are not limited to, obesity;diabetes; other metabolic conditions and diseases; liver diseases andcardiovascular diseases. Because obesity and diabetes are associatedwith an increased risk of several cancers, treatment of obesity anddiabetes is expected to have a beneficial effect on these cancers.

In some embodiments, a bitter taste receptor ligand, and in particularbitter tastants, and/or agent herein described can be comprised in acomposition together with a suitable vehicle. The term “vehicle” as usedherein indicates any of various media acting usually as solvents,carriers, binders or diluents for the bitter taste receptor ligandcomprised in the composition as an active ingredient. Exemplarycompositions comprise PTU, PTC, denatonium benzoate, Glycyrrhizic acidammonium salt, Epigallocatechin gallate, Hyperforin, Berberine chloride,Coptisine Chloride Allyl methyl sulfide, Rottlerin, Curcumin, Ellagicacid, Embelin, a derivative thereof and a combination thereof. In someembodiments, the compositions are formulated for systemic release.

In some embodiments, the ligand or related agents, combination orcomposition is administered by enteral administration. Enteraladministration is a systemic route of administration where the substanceis given via the digestive tract, and includes but is not limited tooral administration, administration by gastric feeding tube,administration by duodenal feeding tube, gastrostomy, enteral nutrition,and rectal administration. In particular in several embodiments, theadministration suitable for these compounds is an oral one in solid orliquid formulation.

In some embodiments, where the composition is to be administered to anindividual the composition can be a pharmaceutical composition, andcomprise za bitter taste receptor ligand and a pharmaceuticallyacceptable vehicle.

In some embodiments, ligand can be included in pharmaceuticalcompositions together with an excipient or diluent. In particular, insome embodiments, pharmaceutical compositions are disclosed whichcontain a ligand, in combination with one or more compatible andpharmaceutically acceptable vehicle, and in particular withpharmaceutically acceptable diluents or excipients.

The term “excipient” as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Suitableexcipients for the pharmaceutical compositions herein disclosed includeany substance that enhances the ability of the body of an individual toabsorb ligand. Suitable excipients also include any substance that canbe used to bulk up formulations with ligand to allow for convenient andaccurate dosage. In addition to their use in the single-dosage quantity,excipients can be used in the manufacturing process to aid in thehandling of the ligand. Depending on the route of administration, andform of medication, different excipients may be used. Exemplaryexcipients include but are not limited to antiadherents, binders,coatings disintegrants, fillers, flavors (such as sweeteners) andcolors, glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluent include any substance that can decrease the viscosityof a medicinal preparation.

Exemplary compositions for enteral administration include but are notlimited to a tablet, a capsule, drops, and suppositories.

In some embodiments, ligands can be administered in combination one withanother and/or with other molecules to modulate one or more hormones.There are several types of combinations that are expected to beeffective. For example, a combination that results in regulation of bothTAS2R38 and TAS2R47 is expected to resulting in release of both CCK andPPY/GLP-1 as will be understood by a skilled person upon reading of thepresent disclosure. In several embodiments, a combination is expected tohave a greater effect than the administration to the single cells. Inparticular, it is expected that combinations of multiple ligands and inparticular bitter tastants can be used to address some conditionsassociated with hormones and in particular, metabolic hormones. Forexample, DB and PTU+PEG are expected to be suitable to treat obesity, astheir administration is expected to result in the release of both CCKand PYY, each of which addresses satiety. This is expected to occur withother hormones released from other GI-mucosa cells in response toactivation by T2R ligands activating a TAS2R receptor on a TAS2R-bearinghormone-secreting cell.

In an embodiment, combination of ligands is expected to provide abeneficial additive effect at an individual endocrine cell type. Forexample, the cell type can include another type of bitter taste GPCR inaddition to TAS2R38 for the L cell or TAS2R47 for the I cell. Examplesof other types of taste receptors are expected to include receptorsresponding to berberine chloride, cyanidine chloride, coptisinechloride.

Suitable schedules of administration that are known or expected to beeffective for the method at issue comprise delivery of agents before andduring a meal as will be understood by a skilled person upon reading ofthe present disclosure.

The Examples section of the present disclosure illustrates examples ofthe compositions and methods herein described as well as the studiescarried out by applicants in order to investigate the functional andphysical interactions of ligands/agents, combination thereof and bittertaste receptors

Further advantages and characteristics of the present disclosure willbecome more apparent hereinafter from the following detailed disclosurein the Examples given by way or illustration only with reference to anexperimental section.

EXAMPLES

The active agents, methods, systems and compositions herein disclosedare further illustrated in the following examples, which are provided byway of illustration and are not intended to be limiting.

Example 1 Methods and Systems to Predict 3D Structures of BTR

The GEnSeMBLE and GenDock methods and systems were used to performprediction of 3D structure of bitter tastant receptors.

Alignment and Homologize. The sequences of TAS2R38 receptors werealigned with the turkey β1 Adrenergic Receptor (thβ1AR) as shown inFIG. 1. Then the residues of hβ1AR were mutated to the aligned sequencesof TAS2R38 receptors and generated the initial 3D structure of TAS2R38receptors.

Optimization of helices. The predicted TM domains of the protein wereextracted to form 7 single helices, which were minimized using thedreiding force field [Mayo 1990] and then merged to form a 7 helixbundles that matches the template.

Construction of a template structure. Given the optimum helices todescribe each of the 7 TM domains, they are placed into a 7-helix bundleusing the x-ray template. Each experimental template has 42 degrees offreedom: x, y, z, θ, φ and η values for each of the seven TM helices(6×7=42 total). The hydrophobic center is the residue that crosses z=0,which is defined as the plane that runs through the center of the lipidbilayer. It is either calculated from the protein's hydrophobic profileor by homology. The degrees of freedom that were optimized are the tiltangle of the helix θ, the sweep angle of the helix φ, and the rotationof the helix η around the helical axis.

Bihelix. After minimization of helices, all possible 7-helix bundlesconstructed were considered by allowing each of the 7 helices to take on12 orientations (30° increments) about their axes, which leads12⁷=35,000,000 packings of the seven helices of the GPCR. The BiHelixprocedure [Goddard 2010] estimates the energies of these 35 millionpackings using a mean field constructed by considering the 12 sets ofnearest neighbor bi-helix interactions, TM1-TM2, TM1-TM7, TM2-TM3,TM2-TM4, TM2-7, TM3-TM4, TM3-TM5, TM3-TM6, TM3-TM7, TM4-TM5, TM5-TM6 andTM6-TM7. Here SCREAM was used to optimize the side-chains for each case[Kam 2008].

CombiHelix. Next, a 7-helix bundle were built into the best 1000 resultsfrom BiHelix and re-optimize the side-chains using SCREAM. Each bundleis also immersed in an implicit membrane to compute membrane solvationeffects that should disfavor helix rotations that expose chargedresidues to lipids. This membrane solvation is described in [Goddard2010].

SuperBiHelix. For the optimum set of rotation angles (η) from step E,now a range of tilts (θ, φ) was sampled simultaneous with to obtain theoptimum 7-helix bundles. Again the 12 pairs of strongly interactinghelices were considered but to account for the effect of tilts. Theseven-helix bundle is partitioned into three quadhelix bundles, as shownin FIG. 1. The 2000 structures with the lowest energy for each quadhelixare selected by increasing energy. Finally, from each individual helicalconformation list, the best 36 conformations for each helix are used tocalculate the energy of 36⁷≈8×10¹⁰ full bundles, and output the 1000combinations estimated from this procedure to have the lowest energies.

SuperComBiHelix. These top 1000 helical bundles from SuperBiHelix arebuilt into 7-helix bundles and the side chains are reoptimized. Then thestructure is minimized for 10 steps. This procedure results in anensemble of low-lying bundles. Examination of the low-lying structuresshows which helices are flexible, and may give insight into activation.

Prediction of the extracelluar (EC) and intracelluar (IC) loopstructure. To provide initial structures for the three EC and IC loopsfor use in MD studies of the bitter taste receptors, the alignment ofbitter taste receptor with tβ1AR were used to homology threaded theseloops to the crystal structure. Then, minimization and dynamics werecarried out on the loops with fixed helix bundle atoms. It was expectedthat these loops to be quite flexible and strongly affected by thesolvent, which was treated only implicitly in the previous steps.

Example 2 Methods and Systems to Identify BTR Ligands Binding Sites

The following methods and systems were used to identify of dockingligands and predicted protein structures.

The structures of the BTR with top 10 total energy identified using werechosen from SuperBiHelix step each of which was used to dock agonistsPTC and PTU.

The structure and charges of PTC and PTU were calculated using quantummechanics (B3LYP with the 6-311G** basis set). Two PTC conformers andfour PTU conformers were used for docking. The GenDock general procedurefor docking was used.

The GenDock method was used to select ligand-binding conformations andcalculating their binding energies. The whole protein was partitionedinto 32 regions (each with sides of 10 Å) and scanned to find theputative binding regions (with the six hydrophobic residues, I, L, V, F,Y, and W alanized). This ScanBindSite procedure used DOCK4.0 [Kuntz1982] to generate 1000 conformations in each of these putative regions,selecting the optimum regions based on a combination of burial score andbinding energy. These optimum regions were combined and 10,000 poseswere generated using DOCK4.0, which were scored using the DREIDING 2 FF[Mayo 1990]. The top 1000 (by energy) were de-alanized, SCREAMed, andthen minimized using the DREIDING 3 FF. Then the top 1% (10) wasselected for further minimization of the binding site complex (using theunified binding site including all residues within 4 Å of any of the 10binding poses). The protein and ligand were then neutralized bytransferring protons appropriately in salt bridges and protonating ordeprotonating exposed side-chains (this leading to more reliable energycomparisons) [Bray 2008]. Then the final docked structure with the bestbinding energy was selected.

Molecular Dynamics Simulation. Since the description of lipid and waterin BiHelix is implicit with a skimpy layer of lipid bilayer, moleculardynamics (MD) simulations of the predicted structure of bitter tastereceptor were performed with and without ligand for 10 ns in explicitlipid bilayer and water. MD simulations using NAMD [Phillips 2005]including explicit water and a periodically infinite lipid were carriedout to determine the interactions of the protein with lipid and water.The predicted protein structure was stripped away the lipid molecules,and inserted in a periodic structure of1-palmytoil-2-oleoylsn-glycero-3-phosphatidylcholine (POPC). In thisprocess, lipid molecules were eliminated within 5 Å of the protein.Then, this was inserted in a box of water molecules and eliminatedwaters within 5 Å of the lipid and protein. Chloride ions were added toneutralize the charge of the system. The membrane and water moleculeswere minimized with the protein fixed, and then equilibrated for 500 psin an NPT simulation. Finally, the entire system was minimized, and then10 ns of NPT simulation was run. All NPT simulations were run usingLangevin dynamics with a damping coefficient of 1 ps⁻¹ and a bathtemperature of 310K. The pressure was kept constant by Nosé-HooverLangevin piston pressure control, with a target pressure of 1 atm andbarostat oscillation and damping times of 200 fs. The stepsize was 1 fs,with periodic boundary conditions applied. The full system (FIG. 3)contains the predicted protein, 101 lipid molecules, 7528 watermolecules, and 19 chlorine ions for a total of 41570 atoms per periodiccell. The box size is 75 Å by 75 Å by 85 Å. The NAMD program was thenused to carry out 10 ns of NPT MD with a bath temperature of 310K.

Example 3 Structure Prediction of hTAS2R38 Bitter Receptor in Humans

Applicants have carried out structure predictions of the human PAV andAVI TAS2R38 receptors. FIG. 21 shows predicted results of thetransmembrane (TM) regions for this receptor in humans and rats.Applicants used these TM regions to generate optimized helices (TMs 5,6, and 7 show bends near their Prolines).

In predicting the structure of the PAV and AVI human T2R38 proteins,Applicants found that TM7 of AVI is rotated 30 degrees to that of PAV,such that the bulkier residue, I, is oriented more towards the exteriorof the protein. All other TMs display the same rotations. The PAVprotein is stabilized by 4 interhelical hydrogen bonds:

a. T32 of TM1 with H68 of TM2 (2.01 Ang.); Q77 of TM2 with Q104 of TM3(1.85 Ang.);b. Q77 of TM2 with W108 of TM3 (2.01 Ang.); R138 of TM3 with H126 of TM4(1.64 Ang.).

The AVI protein is stabilized by 2 interhelical hydrogen bonds:

a. Q77 of TM2 with Q104 of TM3 (1.86 Ang.); Q77 of TM2 with W108 of TM3(2.02 Ang.)

Preliminary docking simulations of PTC to both PAV and AVI result in PTCbinding more strongly to PAV by ˜1.6 kcal/mol. PTC forms a 1.84 Anghydrogen bond with E25 in TM1 of PAV but not in AVI.

Example 4 Modeling the 3D Structure TAS2R38 Bitter Receptor andComparison of the 3D Structure of Various Polymorphisms of TAS2R38Bitter Receptors

Applicants constructed the structural models of the TAS2R38 bitterreceptors (four haplotypes hTAS2R38_(PAV), hTAS2R38_(AVI),hTAS2R38_(AAI) and hTAS2R38_(PVV)) using the turkey β1 AdrenergicReceptor (tβ1AR) as a template based on the homology method.

Applicants shortened or lengthened the tβ1AR TMs to fit the TMpredictions obtained by homologize technology. After minimization, theinitial structures that combine the seven TM segments from tβ1AR(without loops and the eighth helices) are built. The ensemble of a fewthousands conformations with different helical rotation angles η, tiltangle θ and sweeping angle φ were generated based on Bihelix andSuperBihelix technology described in [Goddard 2010].

FIG. 1 shows the Transmembranes (TMs) sequence alignments betweenhTAS2R38_(PAV), hTAS2R38_(AVI), hTAS2R38_(AAI), hTAS2R38_(PVV) and hβ1ARwhich are used to build the 3D structures by the homology modeling. Asshown in Table 1, BiHelix results suggest that the helices of fourvariants have identical rotational angles except helix 6.

TABLE 1 ComBiHelix results for bitter taste receptors in the β1adrenergic receptor template. Receptor Rotational Angle Total EnergyVariants H1 H2 H3 H4 H5 H6 H7 (Kcal/mol) hTAS2R38_(PAV) 30 330 60 90 180270 30 598.1 hTAS2R38_(AVI) 30 330 60 90 330 270 30 668.9 hTAS2R38_(AAI)30 330 60 90 240 270 30 584.1 hTAS2R38_(PVV) 30 330 60 90 240 270 30597.4

The TAS2R38 bitter receptors, class C GPCRs, lack some of thewell-conserved motifs present in class A GPCRs. Thus it can be expectedthat the TAS2R38 bitter receptors might have a different set ofstabilizing interhelical hydrogen bonds from hβ1AR. The predicted 3Dstructures of four variants of the TAS2R38 bitter receptors are shown inFIG. 4, and the residues forming interhelical H-bonds are highlighted.

Applicants find the interhelical hydrogen bonds between Y199 (5) andW108 (3), and between Y199 (5) and A262 (6) in hTAS2R38_(PAV) protein.An interhelical bond between W108 (3) and A(V)262 (6) exists in bothhTAS2R38_(AAI) and hTAS2R38_(PVV) protein. These interhelical bondsdon't exist in the hTAS2R38_(AVI) protein.

To address the 3D structures of these four variants, Applicantsperformed 10 ns of molecular dynamics simulation on the hTAS2R38_(PAV)and hTAS2R38_(AVI) protein structures.

FIG. 5 shows the 3D structures of the hTAS2R38_(PAV) and hTAS2R38_(AVI)proteins after 10 ns MD with lipid and water. The hydrogen bonds inhTAS2R38_(PAV) protein were broken, and two new hydrogen bonds betweenY199 (5) and W108 (3), and between W108 (3) and A262 (6) which is sameto that in hTAS2R38_(AAI) and hTAS2R38_(PVV) protein. We also find 2 newinterhelical hydrogen bonds between W108 (3) and A261 (6), and betweenY199 (5) and A266 (6) in hTAS2R38_(AVI) protein. FIG. 6 shows that thesenew H-bonds are all maintained during the 10 ns MD.

FIG. 7 shows the root mean square deviation (rmsd) time evolution of thehelical segments during the 10 ns MD. Here rmsd is with respect to thelast frame of the 10 ns trajectory. The total rmsd's range from theinitial predicted structure to the final one in the trajectory rangesfor 0.5-2.5 {acute over (Å)} in hTAS2R38_(PAV) and for 1.0-7.0 {acuteover (Å)} in hTAS2R38_(AVI). Focusing on the last 2 ns, thesefluctuations range from 0.5 to 1.5 {acute over (Å)}, except TM2 inhTAS2R38_(PAV) which reaches 2.0 {acute over (Å)}.

Example 5 Modeling Binding Sites of TAS2R38 Bitter Receptors for AgonistPTC and PTU

Applicants used the GenDOCK techniques to predict the binding site ofagonists to the four predicted (lowest energy) structures of TAS2R38bitter receptors. The predicted binding sites of agonist PTC and PTU inhTAS2R38_(PAV) and hTAS2R38_(AVI) protein are shown in FIG. 8. PTC andPTU located between the TM 3, 5 and 6 helices. The most importantresidues (cavity analysis) are (including the interaction energy withligands in parentheses) stated as follows:

a. Xx hTAS2R38_(PAV) with PTC: ALA262 (−5.336), TYR199 (−5.209), CYS112(−2.68), TRP108 (−2.579), LEU 109 (−2.564), ILE 265 (−1.235), PRO 204(−1.106), SER 266 (−0.976).b. hTAS2R38_(PAV) with PTU: ALA262 (−4.085), TYR199 (−2.975), TRP108(−2.127), CYS112 (−1.746), VAL203 (−1.699), LEU109 (−1.655), ALA263(−1.155), PRO204 (−1.056).c. hTAS2R38_(AVI) with PTC: ALA105 (−4.637), TRP108 (−3.035), CYS198(−2.410), ILE265 (−1.982), LEU109 (−1.937), VAL262 (−1.525), SER202(−1.289), SER266 (−0.999).d. hTAS2R38_(AVI) with PTU: SER266 (−4.047), GLN104 (−3.994), CYS198(−3.075), TRP108 (−3.072), ILE265 (−2.243), VAL262 (−2.032), LEU109(−1.965), ALA 105 (−1.149).

As shown in FIG. 9, there are two strong hydrogen bonds between PTC andALA262 in hTAS2R38_(PAV), and the π-π interaction between PTC and TYR199in hTAS2R38_(PAV). There is a hydrogen bond between PTU and ALA262 inhTAS2R38_(PAV). In hTAS2R38_(AVI), PTC interacts with ALA105 by H-bond,while PTU interacts with SER266 and GLN104 by H-bonds. Furthermore, PTCand PTU were docked to other tasters hTAS2R38_(AAI) and hTAS2R38_(PVV).These results suggest that the residue 262 is very important to tastebitter which agrees with experimental data as reported by [Bufe 2005].

After inserting the predicted hTAS2R38_(PAV)/PTC and hTAS2R38_(AVI)/PTCcomplexes into the infinite lipid membrane and solvating fully withwater (using the procedure described in section 2.3), Applicantsperformed 10 ns of MD. FIG. 9 a compares the structure of thehTAS2R38_(PAV)/PTC complex after 10 ns molecular dynamics with theinitial predicted structure. Hydrogen bonds of the original predictedstructures were found to remain stable during 10 ns dynamics. However,one additional hydrogen bond is formed between —NH₂ group of PTC andY199 (5) in hTAS2R38_(PAV). FIG. 10 shows the time evolution for thehydrogen bonds distances between PTC and hTAS2R38_(PAV).

i. —NH—A262 (6): the hydrogen bond distance remains mostly between 1.7and 3.0 Å except the occasional extensions to 4.9 Å during initial 10ps.ii. —NH₂—Y199 (5): the initial distance is 6.6 Å, but it quicklycontracts to 3.0 Å and then mostly fluctuates between 2.0 Å and 4.0 Åand finally come back to ˜2.5 Å.

Thus, Applicants conclude that PTC forms strong interactions with bothTM5 and TM6 in the hTAS2R38_(PAV) structure. The binding of ligandbetween TM5 and TM6 breaks the strong coupling between TM3 and TM6 andbetween TM3 and TM5.

As shown in FIG. 9 b, the ligand in the structure of thehTAS2R38_(AVI)/PTC complex after 10 ns molecular dynamics moves up,compared with that of the initial predicted structure. So the initialH-bond between ligand and A105 was broken, and a water molecule movesinto the binding site and form H-bonds with the ligand. The insertion ofligand in hTAS2R38_(AVI) protein breaks interhelical hydrogen bondsbetween W108 (3) and A261 (6), while not that between Y199 (5) and A266(6).

Example 6 Activation of TAS2R38 Bitter-Taste Receptor

The protein structure prediction and MD simulation results indicate thatthe H-bonds between W108 (3) and A(or V)262 (6) is expected to stabilizethe taster hTAS2R38_(PAV), hTAS2R38_(AAI) and hTAS2R38_(PVV), and thatthe H-bonds between W108 (3) and A261 (6) stabilize the non-tasterhTAS2R38_(AVI). Applicants furthermore find that both PTC and PTU caninteract with A(or V)262 in taster (hTAS2R38_(PAV), hTAS2R38_(AAI) andhTAS2R38_(PVV)) by hydrogen bond, while there is not any H-bond betweenligands and non-taster (hTAS2R38_(AVI)). Applicants outline here thedifference between the agonist-bound taster and agonist-boundnon-taster. The major difference involves the position 262 in the bittertaste receptors. The residue 262 is involved in not only the TM3-TM6interaction but also the binding of agonist to receptors.

Fluorescence experiments on rhodopsin during activation show thatTM3-TM6 interaction is involved in GPCR activation [Farrens 1996]. Themutation experiments indicate that position 262 is important to tastebitter for the TAS2R38 bitter receptors [Bufe 2005]. Accordingly, theresults indicate that the H-bonds between W108 (3) and A(or V)262 (6)and between agonists and A(or V)262(6) in taster play a crucial role inGPCR activation and bitter-tasting, which is consistent with theexperimental data [Bufe 2005].

As shown in Table 1, in addition, the mutation of A262V and V296Iresults in a larger rotation angle of TM5 in hTAS2R38_(AVI) and make theY199 closer to TM6 so that the formation of the hydrogen bond betweenY199 and A266 could lead to the form of H-bond between W108 and A261 notV262. Although the mutation of A262V exists in the hTAS2R38_(PVV), thesmaller rotation angle of TM5 cannot cause the formation of TM5-TM6hydrogen bond interaction. Thus the hydrogen bond interaction betweenTM3 and TM6 may pass the signal to intracellular to activate receptor.

Importantly, both PTC and PTU have H-bonds with residue 262 of thetasters (hTAS2R38_(PAV), hTAS2R38_(AAI) and hTAS2R38_(PVV)) and not withthat of nontaster (hTAS2R38_(AVI)). MD simulation results suggest thatPTC forms the stable H-bonds with Y199 in TM5 and A262 in TM6 ofhTAS2R38_(PAV) structure, while PTC in hTAS2R38_(AVI) prefers to moveaway from V262. So the residue 262, involved in the interaction betweenPTC and bitter taster structure, is very important to taste bittercompounds, which agrees with the literature [Bufe 2005].

The 3D structures of four haplotypes of TAS2R38 G-protein coupledreceptors (GPCRs) were predicted to understand the bitter tastereceptors. Applicants find the formation of H-bonds between W108 (3) andA262 (6) in hTAS2R3_(8PAV) (taster) and between W108 (3) and A261 (6) inhTAS2R3_(8AVI) (non-tatser) could stabilize the protein structure. Tofurther validate these structures, Applicants used the GenDock method topredict the binding sites and 3D structures for PTC and PTU bound tohTAS2R3_(8PAV), hTAS2R3_(8AVI), hTAS2R3_(8AAI) and hTAS2R3_(8PVV),respectively. The predicted binding sites position PTC and PTU in theregion between TM3, TM5 and TM6. The PTC and PTU could form the H-bondswith residue 262 in the tasters (hTAS2R3_(8PAV), hTAS2R3_(8AAI) andhTAS2R3_(8PVV)) and not with that in non-taster (hTAS2R3_(8AVI)). TheH-bonds between PTC and A262 (6) and between PTC and Y199 inhTAS2R3_(8PAV) are stable. However, PTC in hTAS2R3_(8AVI) moves up awayfrom V262 (6) during 10 ns MD. As a consequence, the results indicatethat the hydrogen bond interaction between TM3 and TM6 may pass thesignal to intracellular to activate receptor and that the H-bond betweenagonists and residue 262 in tasters is involved in the bitter tasting,which agrees with the experiment result (Bufe, et al., 2005).

Example 7 Modeling Human TAS2R47 (hTAS2R47) Bitter Taste Receptor

Applicants have obtained a preliminary 3-D structure for hTAS2R47complexed to a family of ligands tested in a study by [Pronin 2004].These results are presented below, along with structure predictionresults for another GPCR (human prostaglandin DP) that providesvalidation of the methods.

Applicants used Membstruk to obtain a 3-D structure for hTAS2R47 andused MSCDock to predict the binding sites for four ligands to hTAS2R47:Denatonium (DD), DD2 (Denatonium derivative), 4-Nitro saccharin (4NS),and 6-Nitro saccharin (6NS). These ligands had been used by [Pronin2004] to study the activation of hTAS2R47, 43, and 44.

FIG. 19 shows predicted TM helix domains for hTAS2R47 and the predictedTM helix bundle structure along with predicted binding regions used fordocking the four ligands. The visible bend in TM7 helix occurs at theposition of the middle Proline in TM7 sequence.

FIG. 20 shows the predicted binding sites for the four ligands. Proninet al. [Pronin 2004] had observed that hTAS2R47 was activated strongestby Denatonium, whereas DD2 and 6NS activated the receptor to a lesserextent and 4NS acted like a very weak inverse agonist. Applicants findthat Denatonium leads to the largest predicted binding energies whilethe other ligands (DD2, 4NS, 6NS) bind with at least 25% weaker bindingenergies compared to Denatonium. These results are consistent with thoseof Pronin et al. [Pronin 2004], assuming that there is a correlationbetween experimental activation rates and computed binding energies. Thepredicted interaction of DD with Asp150(TM4) is consistent withmutagenesis studies. The predicted ligand: hTAS2R47 complexes suggestmultiple binding sites and multiple binding modes for different ligands.

Example 8 Structure of Prostaglandin DP Receptor and Antagonist BindingSites

Applicants used the previous generation MembStruk method to predict thestructure of the DP prostaglandin receptor and then used this predictedstructure with HierDock to predict the binding sites for four familiesof ligands. This work, summarized below, shows that the predicted GPCRstructures are sufficiently accurate for use in drug development.

The predicted binding mode of the lead compound is shown in FIG. 22,which provides an explanation of the important interaction of ligand awith the receptor: i) The methoxy on benzene ring A interacts withR310(7), ii) Benzene ring A and pyrimidine ring are located in ahydrophobic cavity that includes L26(1), K76(2), F115(3), S316(7),L312(7), and 1315(7), iii) The conserved signature residue D72(2) formsa H-bond with the proton of the HN—CH2-C(CH3)2 part of the ligand, iv)Benzene ring B is located among TM127, interacting with N34(1), L68(2),T62(2), and D319(7). v) O-Methyl on benzene ring B forms an HB withT62(2).

Based on the predicted binding mode, Applicants predicted the bindingenergies of many modified compounds. Eight of these cases shown in FIG.23 had been tested experimentally and there was perfect agreementbetween predicted relative binding energies and relative bindingconstants [Li 2007].

Compound b has modifications on ring B and the linker HN—CH2-C(CH3)2,leading to better interactions with S316(7) and D319(7), whichApplicants predicted would improve the binding energy by 1.4 kcal/mol.Indeed Sanofi-Aventis measured IC50=104 nM for b, an improvement by afactor of 8.

Compounds c and d involve modifications on ring B. Compound d removes aCH2 from the linking, reducing the predicted binding by 1.7 kcal/mol,due to repulsive van der Waals contacts. This was confirmedexperimentally, with an increase of IC50 to 1073 nM. Compound c replacesthe benzene with thiophene, leading to a predicted improvement of 3kcal/mol and a measured improvement in IC50 by a factor of 78.

Compounds e to i have modifications on ring A. Substitutions withcarbonic acid or other similar groups were predicted to lead tosubstantial improvements in binding due to favorable interaction withR310(7). These compounds were also found experimentally to have improvedbinding affinity (IC50) as shown in FIG. 23. Indeed the best compound ifrom the theory has the best-observed binding, with IC50=0.8 nM, 1000times better than the starting compound a. In addition, the compoundspredicted to be 2^(nd) best, 3^(rd) best etc. are in exactly the samesequence as in the experiments.

Example 9 Exemplary PTU Derivatives

For a sustained activation of bitter taste receptors in the GI system itis desirable that the bitter receptor ligand (e.g., a PTU derivative)remain in the GI system and not get absorbed. This requires ligands withaltered pharmacokinetic properties like absorption, distribution,metabolism, excretion and toxicology.

Based on the PTU binding site in hTAS2R38_(PAV) (FIG. 8), it wasobserved that the propyl group is exposed and not interacting with thereceptor. This suggests that this functional group can be potentiallyused to attach to complementary molecules like amino acids, peptides,polyethylene glycols (PEGs), or saccharides.

Shown in FIG. 11 is a chemical formula of PTU (C₇H₁₀N₂₀S), the sevencarbon atoms are denoted by the numbers 1 through 7. The sulfur,nitrogen and oxygen atoms are denoted S, N, and O, respectively. Carbonatoms 1, 2, and 3 form a chain that attaches to carbon atom 4. Carbonatoms 2 and 3 each have two hydrogen atoms and carbon atom 1 has threehydrogen atoms. One of the these three hydrogen atoms can be replaced byan OH functional group or the propyl group can be functionalized with acarboxylic acid or azide group to form a covalent bond to an amino acid,peptide, PEGs, monosaccharide, oligosaccharide or other suitablecomplementary molecules.

Shown in FIG. 12 is PTU attached to an arbitrary R functional groupthrough a linker of length n, where n can take a value in the range1-20. This attachment is practically facilitated by firstfunctionalizing PTU with reactive groups like carboxylic acid and azide(FIG. 13). These reactive groups enable attaching large molecules likecellulose and PEGs (FIG. 14) to PTU to produce PTU derivatives withdesirable pharmacokinetic properties.

It has been shown that intestinal permeability of PEGs is highlydependent on their molecular weight [Kerckhoffs 2010]. This provides alower limit of 10,000 for PEG's molecular weight, which will keep, e.g.,PTU coupled to this PEG, in the GI system for a prolonged activation ofbitter taste receptors in the gut resulting in a sustained modulation ofthe hormone release.

Example 10 Preparation of Active Agent Conjugated with a ComplementaryMolecule: PTU-Cellulose

An active agent conjugated with a suitable complementary molecule can beprepared using known methods. An exemplary reaction scheme forconjugation with a suitable complementary molecule. In the example belowshows chemical synthesis of PTU-cellulose.

One of PTU's pharmacokinetic properties (distribution) was modifiedthrough conjugation to cellulose. This and other modifications canmodify PTU's full pharmacokinetic profile (Absorption, Distribution,Metabolism, Excretion, and Toxicology).

Example 11 PTC Administration Results in an Increased Release of PYY

Applicants administered either 5 ml of saline or 5 mM PTC in 5 ml ofsaline into the stomachs of fasting conscious male Sprague-Dawley rats(250-300 gm in weight) by standard gastric gavage. The rats were theneuthanized 45 min later and removed blood via intracardiac puncture andmeasured PYY in the blood by a recently modified radioimmunoassay usingCURE antibody 9153 (Gift of J Reeve, University of California, LosAngeles.). Results demonstrated a doubling of PYY levels as follows:Saline gavaged rats (n=5) 71+/−35 pM, PTC gavaged rats (n=5) 154+/−44pM. An interesting observation at time of sacrifice of the rats was thatthe volume of fluid remaining in the stomachs of the PTC-treated ratsranged from 1-2 ml whereas there was less than 0.5 ml in the stomach ofsaline-treated rats. This observation suggests that PTC throughinteraction with TAS2R38 regulates gastric emptying.

Example 12 PTC Administration Results in GLP-1 Release in EndocrineCells

In order to determine the feasibility of a cell-based system forinvestigating the selectivity of ligands for a particular bitter tastereceptor, Applicants chose STC-5 cells and measured the effect of PTC onGLP-1 from these cells. Applicants have demonstrated that STC-5 cellshave TAS2R38 among several other bitter taste receptors.

In GLP-1 release experiments, selected endocrine cell lines of STC-5cells or NCI-H716 cells were used for testing release of GLP-1 triggeredby various possible ligands. Endocrine cells were incubated inserum-free DME/F12 media containing vehicle (0.01% DMSO) and a candidateligand of certain concentration and for a certain period of incubationtime. After incubation, secretion active GLP-1 in media was measuredwith GLP-1 RIA kit (Linco Research®, Missouri, USA).

In particular, STC-5 cells were incubated for 30 minutes in serum-freeDME/F12 media containing vehicle (0.01% DMSO) and PTC at the indicatedconcentrations. Secretion of active GLP-1 in media was measured withGLP-1 RIA kit (Linco Research, Missouri, USA). The experiments for allgroups have been conducted in duplicates.

The results illustrated in FIGS. 24A and 24B show that PTC had a largeeffect on GLP-1 release from these cells, while the vehicle (negativecontrol or PTC concentration of 0) does not. This shows that bitterligands like GLP-1 can be effectively used for the release of GLP-1.

Example 13 Glucagon-Like Peptide-1 (GLP-1) Release from Endocrine CellLine, NCI-H716 Cells

Endocrine cell lines of NCI-H716 cells were used for testing release ofGLP-1 triggered by various possible ligands.

NCI-H716 cells were incubated in serum-free DME/F12 media containingvehicle (0.01% DMSO) with a candidate ligand at the indicatedconcentrations. Secretion active GLP-1 in media was measured with GLP-1RIA kit (Linco Research, Missouri, USA). The results were summarized inTable 2. The experiments for all groups have been conducted induplicates.

TABLE 2 GLP-1 release by NCI-H716 cells triggered by various ligands.GLP-1 (pmol/mg Groups Concentration protein) Control 0 58.47 PTC 2.5 μm98.73 Butyrate 2.5 mM 100.77 Glycyrrhizic acid ammonium salt 10 μm 71.95Epigallocatechin gallate 1 μm 91.15 Ginseng Root 10 μm 62.21 Hyperforin5 μg/ml 104.87 Berberine chloride 10 μm 86.41 Coptisine chloride 10 μm121.35 Allyl methyl sulfide 10 μm 91.547 Hops (Hopsteiner BetaBio45) 10μm 86.149 Rottlerin 0.05% 111.18 Curcumin 10 μm 61.52 Ellagic acid 10 μm77.25 Embelin 10 μm 83.55

PTRU, Epigallocatechin and Berberine choloride are exemplary bittertastant receptor ligands that are also bitter tastants. Hopsteiner is anexemplary combination of ligands which results in a bitter tastantscombination. Ginseng root is an exemplary combination of bitter tastantreceptor ligands including many, many different components. Theremaining ligands are not bitter tastants. The results illustrated inTable 2 show that PTC, butyrate, glycyrrhizic acid, epigallocatechingallate, hyperforin, berberine, coptisine, ally sulfide, the B-acids inhops (Hopsteiner BetaBio45 composition), rottlerin, ellagic acid andembelin each increase the release of GLP-1 from NCI-H716 cells, a modelcell line representing the L-cell.

Examples 14 Endocrine Cells Presenting Bitter Tastant Receptor andRelated Hormones

Exemplary endocrine cells expected or known to present bitter tastantreceptor ligands are listed in the following Table 3 together with thespecific metabolic hormones associated with each of those different GIcells.

TABLE 3 Types of enteroendocrine cells and their secreted products CellType Secreted hormone Location α cells Glucagon Pancreas (Islets ofLangerhans) β cells Insulin, Islet Pancreas (Islets of Langerhans)amyloid, polypeptide PP cells Pancreatic polypeptide Pancreas (Islets ofLangerhans) δcells (D cell) Somatostatin Pancreas (Islets of Langerhans)cells Gastrin Stomach-Occasionally in pancreas X/A-like cells Ghrelin,nesfatin-1 Stomach-Occasionally in pancreas GIP cells (K cells) GIP,xenin Small intestine S cells Secretin Small intestine I cells (CCKcells) Cholecystokinin Small intestine N cells Neurotensin Smallintestine L cells PYY, GLP-1, GLP- Small and large intestine 2,oxyntomodulin Abbreviations: CCK, cholecystokinin; GIP, gastricinhibitory polypeptide; GLP, glucagon-like peptide; PPY, peptide YY.From Field, B. C. T. et al. Bowels control brain: gut hormones andobesity Nat. Rev. Endocrinal, 2010; 6:444-453.

Example 15 Selection of Complementary Molecule for a Certain Ligand

In order to select an appropriate complementary molecule to be tetheredto a determined ligand, it is expected that the ligand can be tetheredto a molecule of a size that has a minimum weight determined withreference to the existing literature. One skilled in the art would knowor be able to find in the literature that certain molecular weights ofPEG will be unlikely to leave the lumen of the intestine. Thus, thosemolecular weights and greater are those one would use for acomplementary molecule to be conjugated to ligands intended to bind GIbitter taste receptors and not to be absorbed across the lumen of theintestine.

In one aspect, one skilled in the art could consult a study (Kerckhoffs)of IBS patients (and comparing to healthy patients) testing molecularmasses M (sub r) of 400, 1500, 4000 and 10,000 and showing that undercertain conditions the 10,000 weight PEG migrated out of the intestine(present in urine) in 7%-33% of the (small) study population. It is notclear that a 10,000 weight PEG would escape in a non-IBS patient,suggesting that for that population lower weight might suffice, but itis possible that for both IBS and non-IBS groups, a complementary PEG ofa greater weight would likely be preferable.

Accordingly, the molecular weight of a molecule intended to keep theagent in the intestine here it is expected to be in the range of 10,000or greater. In this case the tethered molecule, whether PEG or other,and the tether, are expected to both have to be resistant or imperviousto degradation by the digestive enzymes or other chemicals and action ofthe stomach and intestine. The tether would have to be connected to theagent in a way and/or at such a point on the agent that it does notinterfere with/inhibit the agent's functioning/binding. Modification byattaching to cellulose or PEG is expected to accomplish this effect.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the arrangements, devices, ligands, agents,compositions, systems and methods of the disclosure, and are notintended to limit the scope of what the inventors regard as theirdisclosure. All patents and publications mentioned in the specificationare indicative of the levels of skill of those skilled in the art towhich the disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed Thus, it should be understood that although thedisclosure has been specifically disclosed by preferred embodiments,exemplary embodiments and optional features, modification and variationof the concepts herein disclosed can be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. The term “plurality” includestwo or more referents unless the content clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified can beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein can be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably canbe practiced in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the disclosure and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe devices, device components, methods steps set forth in the presentdescription. As will be obvious to one of skill in the art, methods anddevices useful for the present methods can include a large number ofoptional composition and processing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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What is claimed is:
 1. A method for modulating release of a metabolichormone in an individual, the method comprising administering to theindividual one or more GI bitter taste receptor ligands selected fromPTU, PTC, denatonium benzoate, Glycyrrhizic acid ammonium salt,Epigallocatechin gallate, Hyperforin, Berberine chloride, CoptisineChloride, Allyl methyl sulfide, Rottlerin, Curcumin, Ellagic acid,Embelin and/or or a derivative thereof, in an effective amount to allowbinding of the one or more GI bitter taste receptor ligands to the oneor more GI bitter taste receptors in the individual, the bindingresulting in modulating release of metabolic hormone GLP-1, PYY and/orCCK.
 2. The method of claim 1, wherein the one or more GI bitter tastereceptor ligands comprise PTU, PTC, denatonium benzoate, Glycyrrhizicacid ammonium salt, Epigallocatechin gallate, Hyperforin, Berberinechloride, Coptisine Chloride, Allyl methyl sulfide, Rottlerin, Curcumin,Ellagic acid, and Embelin and/or an agonist derivative thereof and thebinding results in increasing release of GLP-1, PYY and/or CCK.
 3. Themethod of claim 1, wherein the one or more GI bitter taste receptorligands comprise denatonium benzoate or an agonist derivative thereofand the binding results in increasing release of CCK.
 4. The method ofclaim 1, wherein the one or more GI bitter taste receptor ligands areadministered in combination with a complementary molecule configured forinterfering with systemic absorption and release of the one or more GIbitter taste receptor ligands.
 5. The method of claim 4, wherein atleast one of the one or more GI bitter taste receptor ligands isconjugated with the complementary molecule to provide a bitter tastantagent configured to present a portion active with respect to the bindingto the bitter tastant receptor.
 6. The method of claim 4 or 5, whereinthe complementary molecule is cellulose, PEG, monosaccharides,oligosaccharides, amino acids, and/or peptides.
 7. The method of claim1, wherein the administering of the one or more GI bitter taste receptorligands is performed in a composition formulated to minimize systemicabsorption of the one or more GI bitter taste receptor ligands across GIepithelium.
 8. The method of claim 1, wherein the administering isperformed by enteral administration.
 9. A composition for modulatingrelease of a metabolic hormone in an individual, the compositioncomprising one or more GI bitter taste receptor ligands selected fromPTU, PTC, denatonium benzoate, Glycyrrhizic acid ammonium salt,Epigallocatechin gallate, Hyperforin, Berberine chloride, CoptisineChloride, Allyl methyl sulfide, Rottlerin, Curcumin, Ellagic acid,Embelin and/or or a derivative thereof together with a suitable vehicle.10. The composition of claim 9, wherein the one or more GI bitter tastereceptor ligands are comprised in combination with a complementarymolecule configured for interfering with systemic absorption and releaseof the one or more bitter taste receptor ligands.
 11. The composition ofclaim 10, wherein at least one of the one or more GI bitter tastereceptor ligands is conjugated with the complementary molecule toprovide a bitter tastant agent configured to present a portion activewith respect to the binding to the bitter tastant receptor.
 12. Thecomposition of claim 9, wherein the one or more GI bitter taste receptorligands are comprised in an amount of from about 1 micromolar to about1.25 millimolar.
 13. The composition of any one of claims 9 to 12,wherein the composition is a pharmaceutical composition and the suitablevehicle is a pharmaceutically acceptable vehicle.
 14. A system formodulating release of a metabolic hormone in an individual, the systemcomprising at least two bitter taste receptor ligands able to modulateone or more target GI bitter taste receptors for simultaneous, combinedor sequential use in the method of claim
 1. 15. A bitter tastant agentfor modulating a target bitter tastant receptor, the bitter tastantagent comprising a bitter taste receptor ligand conjugated to acomplementary molecule configured for interfering with systemicabsorption and release of the bitter taste receptor ligand, wherein thebitter taste receptor ligand comprises a first portion and a secondportion, the first portion active with respect to the binding of thebitter taste receptor ligand to the bitter taste receptor and the secondportion passive with respect to the binding of the bitter taste receptorligand to the bitter taste receptor, and wherein the complementarymolecule is attached to the second portion of the bitter taste receptorligand to provide the bitter tastant agent configured to present thefirst portion of the bitter taste receptor ligand for binding to thebitter taste receptor agent.
 16. The bitter tastant agent of claim 15,wherein the bitter taste receptor ligand is selected from PTU, PTC,denatonium benzoate, Glycyrrhizic acid ammonium salt, Epigallocatechingallate, Hyperforin, Berberine chloride, Coptisine Chloride Allyl methylsulfide, Rottlerin, Curcumin, Ellagic acid, Embelin and a derivativethereof.
 17. The bitter tastant agent of claim 15, wherein thecomplementary molecule is selected from cellulose, PEG, monosaccharides,oligosaccharides, amino acids, and/or peptides.
 18. A bitter tastantagent comprising a bitter tastant receptor ligand conjugated with acomplementary molecule, wherein the complementary molecule is configuredfor interfering with systemic release of the bitter tastant receptorligand.
 19. A method for activating a target bitter taste receptor in anindividual, the method comprising administering to the individual aneffective amount of one or more bitter tastant agent of claim
 18. 20. Acomposition for activating a target bitter taste receptor in anindividual, the composition comprising one or more bitter tastant agentof claim 18 and a suitable vehicle.
 21. A system for providing a bittertastant agent, the system comprising one or more bitter taste receptorligands and at least one complementary molecule configured forinterfering with systemic release of the one or more bitter tastereceptor ligands, wherein the one or more bitter taste receptor ligandsare capable of being conjugated with the at least one complementarymolecule to provide the bitter tastant agent of claim
 18. 22. A methodfor modulating release of a metabolic hormone and a related biologicalprocess thereof in an individual, the method comprising administering tothe individual an effective amount of one or more agents to activate oneor more bitter taste receptors in the individual.
 23. A method toidentify a biological response associated to activation of a bittertastant receptor in a cell, the method comprising contacting the cellwith one or more bitter tastant receptor ligands selected PTU, PTC,denatonium benzoate, Glycyrrhizic acid ammonium salt, Epigallocatechingallate, Hyperforin, Berberine chloride, Coptisine Chloride Allyl methylsulfide, Rottlerin, Curcumin, Ellagic acid, Embelin and a derivativethereof to allow binding of the bitter tastant receptor ligand to thebitter tastant receptor; and detecting the biological response in thecell following the contacting.
 24. The method of claim 23, furthercomprising comparing the detected biological response to a referencebiological response to characterize the biological response.
 25. Amethod to identify a GI bitter taste receptor ligand capable ofmodulating release of metabolic hormones associated to the GI system,the method comprising identifying a GI bitter taste receptor that isexpressed in a target cell in the GI system; predicting the structure ofthe GI bitter tastant receptor, identifying a candidate ligand thatbinds to the GI bitter tastant receptor based on the predicted GI bittertastant receptor structure, testing the identified candidate ligand in abitter taste receptor activation assay, and testing effect of thecandidate ligand on a modulation of at least one of the metabolichormones associated with the GI system.
 26. The method of claim 25,wherein the candidate ligand is identified through modeling of the GIbitter taste receptor.
 27. The method of claim 26, wherein the modelingis homology modeling.
 28. The method of claim 27, wherein the modelingcomprises predicting structures of interhelical hydrogen bonds.
 29. Amethod to modulate hormone release from a cell, the method comprisinginducing a specific conformation of a GI bitter tastant receptor throughbinding of a GI bitter taste receptor ligand, the specific conformationbeing at least one of a plurality of active conformations of the GIbitter tastant receptor; detecting increase or decrease of hormonerelease from a cell following the inducing; and modulating said increaseor decrease through action of the GI bitter taste receptor ligand. 30.The method of claim 29, wherein the inducing is preceded by identifyingthe GI bitter taste receptor ligand by a method comprising identifying aGI bitter taste receptor that is expressed in a target cell in the GIsystem; predicting the structure of the GI bitter tastant receptor,identifying a candidate ligand that binds to the GI bitter tastantreceptor based on the predicted GI bitter tastant receptor structure,testing the identified candidate ligand in a bitter taste receptoractivation assay, and testing effect of the candidate ligand on amodulation of at least one of the metabolic hormones associated with theGI system.
 31. A method to treat or prevent in an individual a conditionassociated to a metabolic hormone, the method comprising administeringto the individual one or more GI bitter taste receptor ligands selectedfrom PTU, PTC, denatonium benzoate, Glycyrrhizic acid ammonium salt,Epigallocatechin gallate, Hyperforin, Berberine chloride, CoptisineChloride Allyl methyl sulfide, Rottlerin, Curcumin, Ellagic acid, andEmbelin and a derivative thereof, in a therapeutically effective amountto modulate metabolic hormone GLP-1, PYY and/or CCK.
 32. The method ofclaim 31, wherein the one or more GI bitter taste receptor ligands areadministered in combination with a complementary molecule configured forinterfering with systemic absorption and release of the one or more GIbitter taste receptor ligands.
 33. The method of claim 32, wherein atleast one of the one or more GI bitter taste receptor ligands isconjugated with the complementary molecule to provide a bitter tastantagent configured to present a portion active with respect to the bindingto the bitter tastant receptor.
 34. The method of claim 32 or 33,wherein the complementary molecule is cellulose, PEG, monosaccharides,oligosaccharides, amino acids, and/or peptides.
 35. The method of claim31, wherein the administering of the one or more GI bitter tastereceptor ligands is performed in a composition formulated to minimizesystemic absorption of the one or more GI bitter taste receptor ligandsby GI epithelium.
 36. The method of claim 31, wherein the administeringis performed by enteral administration.
 37. The method of claim 31,wherein the therapeutically effective amount is from about 1 micromolarto about 1.25 millimolar.
 38. The method of claim 31, wherein thecondition is obesity, diabetes, liver diseases and/or a cardiovasculardisease.
 39. A system for treating or preventing a condition associatedto a metabolic hormone in an individual, the system comprising at leasttwo bitter taste receptor ligands selected from PTU, PTC, denatoniumbenzoate, Glycyrrhizic acid ammonium salt, Epigallocatechin gallate,Hyperforin, Berberine chloride, Coptisine Chloride Allyl methyl sulfide,Rottlerin, Curcumin, Ellagic acid, Embelin and a derivative thereof forsimultaneous, combined or sequential use in the method of claim 31.